Chapter 34, Microbial Lectins: Hemagglutinins, Adhesins ...eskolab.ucsd.edu/advGlyco2016/assigned_reading05-31.pdf · Assigned Reading: Chapter 34, Microbial Lectins: Hemagglutinins,

AssignedReading:Chapter34,MicrobialLectins:Hemagglutinins,Adhesins,andToxins-EssentialsofGlycobiology,2ndeditionManuscript,StructuralEvolutionofGlycanRecognitionbyaFamilyofPotentHIVAntibodiesReview,RoleofReceptorBindingSpecifityinInfluenzaAVirusTransmissionandPathogenesis

Chapter 34 Microbial Lectins: Hemagglutinins,

Adhesins, and Toxins

Essentials of Glycobiology, 2nd edition

3/24/2016 Microbial Lectins: Hemagglutinins, Adhesins, and Toxins - Essentials of Glycobiology - NCBI Bookshelf

http://www.ncbi.nlm.nih.gov/books/NBK1907/ 1/7

Chapter 34 Microbial Lectins: Hemagglutinins, Adhesins, and Toxins

Esko JD, Sharon N.

Many microorganisms exploit host cellsurface glycans as receptors for cell attachment and tissue colonization, and a large number of

pathogenic species depend on these interactions for infection. This chapter describes examples of proteins on the surface of microorganisms

(adhesins or hemagglutinins), secreted proteins (heatlabile toxins), their glycan partners on mammalian cell surfaces (receptors), and

insights into the molecular interactions that take place.

BACKGROUNDViruses, bacteria, and protozoa express an enormous number of glycanbinding proteins or lectins. Many of these microbial lectins were

originally detected based on their ability to aggregate red blood cells (i.e., to induce hemagglutination). The first microbial hemagglutinin

identified was in the influenza virus, and it was shown by Alfred Gottschalk in the early 1950s to bind to erythrocytes and other cells though

sialic acid residues of cellsurface glycoconjugates. Don Wiley and his associates crystallized the viral hemagglutinin and determined its

structure in 1981, and later they solved the structure of cocrystals prepared with sialyllactose. Since then, a number of viral hemagglutinins

have been identified and crystallized. These studies set the stage for analyzing other types of microbial lectins produced by bacteria and

protozoa.

Nathan Sharon and colleagues first described bacterial surface lectins in the 1970s. These lectins also have hemagglutinating activity, but

their primary function is to facilitate attachment or adherence of bacteria to host cells, a prerequisite for bacterial colonization and infection

(see Chapter 39). Thus, bacterial lectins are often called adhesins, and the glycan ligands on the surface of the host cells are called receptors.

Note that the term “receptor” in this case is equivalent to “ligand” for animal cell lectins. Bacteria also produce toxins, whose actions often

depend on glycanbinding subunits that allow the toxin to combine with membrane glycoconjugates and deliver the functionally active

toxic subunit across the plasma membrane. During the last 30 years, many adhesins and toxins have been described, cloned, and

characterized. Additionally, adhesins have been discovered on various parasites. The interactions of adhesins with glycan receptors can help

determine the tropism of the organism.

Colonization of tissues by microorganisms is not always pathogenic. For example, the normal flora of the lower gastrointestinal tract is

determined by appropriate and desirable colonization by beneficial bacteria. The initial events in the formation of nitrogenfixing nodules in

leguminous root tips by species of Rhizobium may also involve lectins on the root tip binding to Nod factors generated by the bacterium

(see Chapter 37).

Many adhesins contain carbohydraterecognition domains (CRDs) that bind to the same carbohydrates as endogenous mammalian lectins

(see Chapter 26). Like animal cell lectins, some microbial adhesins bind to terminal sugar residues, whereas others bind to internal

sequences found in linear or branched oligosaccharide chains. Detailed studies of the specificity of microbial lectins have led to the

identification and synthesis of powerful inhibitors of adhesion that may form the basis for therapeutic agents for treating infection (see

Chapter 51).

VIRAL GLYCAN-BINDING PROTEINSBy far, the beststudied example of a viral glycanbinding protein is the influenza virus hemagglutinin, which binds to sialic acid–containing

glycans. The affinity of this interaction is relatively low, like that of other glycanbinding proteins with their glycan ligands, but the avidity

for cell membranes increases because of oligomerization of the hemagglutinin into trimers and the high density of glycan receptors present

on the host cell (see Chapter 27). Binding is a prerequisite for fusion of the viral envelope with the plasma membrane and for uptake of the

virus into cells. The specificity of the interaction of the hemagglutinin with host glycans varies considerably for different subtypes of the

virus. Human strains of influenzaA and B viruses bind primarily to cells containing Neu5Acα2–6Gal, whereas chicken influenza viruses

bind to Neu5Acα2–3Gal and porcine strains bind to both Neu5Acα2–3Gal and Neu5Acα2–6Galcontaining receptors. This linkage

preference is due to certain amino acid changes in the hemagglutinin. InfluenzaC virus, in contrast, binds exclusively to glycoproteins and

glycolipids containing 9Oacetylated Nacetylneuraminic acid (see Chapter 39).

The specificity of the hemagglutinin correlates with the structures of sialylated glycans expressed on target epithelial cells in animal hosts.

For example, trachea epithelial cells in humans express glycans with a preponderance of Neu5Acα2–6Gal linkages, whereas other tissues



contain many more Neu5Acα2–3Galterminated glycans. Thus, the specificity of the hemagglutinin determines the tropism of the virus with

respect to species and target cells. The hemagglutinin is also the major antigen against which neutralizing antibodies are produced, and

antigenic changes in this protein are in part responsible for new viral outbreaks. In addition, it plays a critical role inside the cell where it

facilitates pHdependent fusion of the viral envelope with endosomal membranes after internalization.

The crystal structure of the hemagglutinin shows two disulfidebonded subunits, HA1 and HA2, which are derived by cleavage of a

precursor protein. The monomer consists of a hydrophilic carboxyterminal domain located inside the viral envelope and a hydrophobic

membranespanning domain. An elongated triplehelical coiled stem region extends 135 Å from the membrane topped by a globular domain

that contains the carbohydraterecognition domain (Figure 34.1). The crystal structure also revealed a hydrophobic pocket near the CRD,

which explains why sialosides containing a hydrophobic aglycone bind the hemagglutinin with greater affinity than simple glycans.

FIGURE 34.1Structure of the influenza virus hemagglutinin (HA) ectodomain. (a) A schematic diagram of the trimeric ectodomain of theH3 avian HA from A/duck/Ukr/63 showing residues HA1 9–326 and HA2 1–172. (Gray, red, blue) Modeled carbohydrateside (more...)

In addition to the hemagglutinin, influenzaA and B virions express a sialidase (traditionally and incorrectly called neuraminidase) that

cleaves sialic acids from glycoconjugates. Its functions may include (1) prevention of viral aggregation by removal of sialic acid residues

from virion envelope glycoproteins, (2) dissociation of newly synthesized virions inside the cell or as they bud from the cell surface, and (3)

desialylation of soluble mucins at sites of infection in order to improve access to membranebound sialic acids. In influenzaC virus, a

single glycoprotein contains both the hemagglutinin activity and the receptordestroying activity, which in this case is an esterase that

cleaves the 9Oacetyl group from acetylated sialic acid receptors. Powerful inhibitors have been designed based on the crystal structure of

the sialidase from influenzaA virus. Some of these inhibit the enzyme activity at nanomolar concentrations and are in clinical use as

antiviral agents (see Chapter 51).

Rotaviruses, the major killer of children worldwide, also can bind to sialic acid residues. These viruses only bind to the intestinal epithelium

of newborn infants during a period that appears to correlate with the expression of specific types and arrangements of sialic acids on

glycoproteins. Many other viruses (e.g., adenovirus, reovirus, Sendai virus, and polyomavirus) also appear to use sialic acids for infection,

and crystal structures are now available for several of their sialic acid–binding domains (Table 34.1).

TABLE 34.1Examples of viral lectins and hemagglutinins

A number of viruses use heparan sulfate proteoglycans as adhesion receptors (e.g., herpes simplex virus [HSV], footandmouth disease

virus, and dengue flavivirus [Table 34.1]). In many cases, the proteoglycans may be part of a coreceptor system in which the

microorganisms make initial contact with a cellsurface proteoglycan and later with another receptor. For example, herpesvirus infection is

thought to involve viral glycoproteins gC and/or gB binding to cellsurface heparan sulfate proteoglycans, followed by binding of viral

glycoprotein gD to one of several cellsurface receptors, including heparan sulfate and one or more members of the Hve (herpesvirus entry)

family of receptors. This step leads to fusion of the viral envelope with the hostcell plasma membrane. The coreceptor role of proteoglycan

is reminiscent of the formation of ternary complexes required for antithrombin inhibition of thrombin and for fibroblast growth factor (FGF)

cell signaling (see Chapter 35). The HSV glycoprotein gB binds heparan sulfate and promotes viruscell fusion and syncytium formation

(cell–cell fusion) as well as adherence. The mechanism by which heparan sulfate facilitates membrane fusion is unknown, but perhaps it

acts like a template facilitating the association of fusogenic membrane proteins of the virus or host cell. Although it is clear that cellsurface

proteoglycans act as adhesion receptors, their role in invasion and pathogenesis has not been established.

Dengue flavivirus, the causative agent of dengue hemorrhagic fever, binds to heparan sulfate. Modeling the primary sequence of the viral

envelope protein on the crystal structure of a related virion envelope protein suggests that the heparan sulfate–binding site may lie along a

groove in the protein lined by positively charged amino acids (Figure 34.2). Thus, the glycosaminoglycan (GAG)binding adhesins may



have a more open structure, consistent with the combining sites of other heparinbinding proteins (see Chapter 35). Footandmouth disease

viruses normally bind to epithelial cells through integrins, but after passage in cell culture, they can acquire the capacity to bind heparan

sulfate. The combining site for heparin is located in a shallow depression formed by three of the major capsid proteins (Figure 34.3). HIV

also can bind to heparan sulfate and other sulfated polysaccharides by way of the V3 loop of the gp120 glycoprotein. Thus, the heparan

sulfatebinding adhesins appear to pick out carbohydrate units within the polysaccharide chains as opposed to binding to terminal sugars.

Interestingly, the interaction of the gD glycoprotein of Herpes simplex virus with heparan sulfate shows specificity for a particular

substructure in heparan sulfate containing a 3Osulfated glucosamine residue, the formation of which is catalyzed by specific isozymes of

the glucosaminyl 3Osulfotransferase gene family (see Chapter 16). However, it is unclear whether other viruses show a preference for

specific heparan sulfate oligosaccharides. A growing body of literature suggests that heparan sulfate and heparin can block the interaction of

the viruses with host cells, suggesting a simple therapeutic approach for treating viral infections.

FIGURE 34.2Two views of a putative heparinbinding site in dengue virus envelope protein. The envelope protein monomer is shown inribbon form, displayed along its longitudinal axis and as an external side view. (Top) Note the alignment of positivelycharged amino (more...)

FIGURE 34.3Model of footandmouth disease virus (FMDV) in complex with heparin trisaccharides. (a) A schematic depiction of theicosahedral capsid of FMDV. VP1–3 contribute to the external features of the capsid: (blue) VP1; (green) VP2; (red) VP3.The (more...)

BACTERIAL ADHESION TO GLYCANSBacterial lectins occur commonly in the form of elongated, submicroscopic, multisubunit protein appendages, known as fimbriae (hairs) or

pili (threads), which interact with glycoprotein and glycolipid receptors on host cells. The best characterized of these are the mannose

specific type1 fimbriae, the galabiosespecific P fimbriae, and the Nacetylglucosaminebinding F17 fimbriae produced by different strains

of Escherichia coli. Fimbriated bacteria express 100–400 of these appendages, which typically have a diameter of 5–7 nm and can extend

hundreds of nanometers in length (Figure 34.4). Thus, pili extend well beyond the bacterial glycocalyx formed from lipopolysaccharide and

capsular polysaccharides (see Chapter 20), which can actually interfere with their activity. The carbohydraterecognition domain of the

fimbriae is found in the minor subunit (FimH of type1 fimbriae, PapG of P fimbriae, and F17G of F17 fimbriae) that is usually located at

the tip of the fimbriae. Some of the other bacterial lectins are monomeric or oligomeric membrane proteins. Most bacteria (and possibly

other microorganisms) have multiple adhesins with different carbohydrate specificities, which help define the range of susceptible tissues

(i.e., the microbe’s ecological niche). Binding is generally of low affinity, but because the adhesins and the receptors often cluster in the

plane of the membrane, the resulting avidity can be great. Perhaps an appropriate analogy for adhesinreceptor binding is the interaction of

the two faces of Velcro™ strips.

FIGURE 34.4E. coli express multiple pili as indicated by the fine filaments surrounding the cells. (Reprinted, with permission of Elsevier,from Sharon N. 2006. Biochim. Biophys. Acta 1760: 527–537; courtesy of David L. Hasty, University of Tennessee,Memphis, (more...)

Examination of the highresolution, threedimensional structure of the glycanbinding subunit FimH of type1 fimbriae in complex with

bound mannose revealed that although mannose exists in solution as a mixture of α and β anomers, only the former was found in the

complex. It is buried at a deep and negatively charged site at the edge of FimH (Figure 34.5). All of the mannose hydroxyl groups, except

the anomeric one, interact extensively with combiningsite residues, almost all of which are situated at the ends of βstrands or in the loops

extending from them. Part of the hydrogenbonding network is identical to that found in mannose complexes of other lectins, such as those

of plants.



FIGURE 34.5αAnomer of mannose in the combining site of FimH. The mannose is buried in a unique site at the tip of the receptorbinding domain (left) in a deep and negatively charged pocket (right). FimH selects the α configuration around the free(more...)

In the urinary tract, the type1 fimbriae of E. coli mediate binding of the bacteria to a protein called uroplakin Ia. This glycoprotein presents

high levels of terminally exposed mannose residues that are capable of specifically interacting with FimH. Anchorage of E. coli to the

urothelial surface via type1 fimbriae–uroplakin Ia interactions may play a role in their colonization of the bladder and eventual ascent

through the ureters against urine flow to invade the kidneys. Another receptor for these fimbriae is the urinary Tamm–Horsfall glycoprotein,

which acts as a soluble inhibitor of the adhesion of the bacteria to the uroplakins and helps to clear the bacteria from the urinary tract.

Indeed, mice lacking the Tamm–Horsfall gene are considerably more susceptible to bladder colonization by type1fimbriated E. coli than

normal mice, whereas they are equally susceptible to Pfimbriated E. coli that do not bind to the same glycoprotein. Type1 fimbriae also are

instrumental in the attachment of E. coli to human polymorphonuclear cells and to human and mouse macrophages. This is often followed

by the ingestion and killing of the bacteria, a phenomenon named lectinophagocytosis, which is an early example of innate immunity (see

Chapter 39).

Other binding specificities have been described as well (Table 34.2). The specificity of binding can explain the tissue tropism of the

organism. The columnar epithelium that lines the large intestine expresses Galα1–4GalCer, whereas the cells lining the small intestine do

not. Thus, Bacterioides, Clostridium, E. coli, and Lactobacillus only colonize the large intestine under normal conditions. Pfimbriated E.

coli as well as different toxins bind specifically to galabiose (Galα1–4Gal) and galabiosecontaining oligosaccharides, most commonly as

constituents of glycolipids. Binding occurs either to terminal nonreducing galabiose units or to internal ones (i.e., when the disaccharide is

capped by other sugars). Pfimbriated E. coli adhere mainly to the upper part of the kidney, where galabiose is abundant. E. coli K99

provides a striking illustration of the fine specificity of bacterial surface lectins and their relationship to the animal tropism of the bacteria.

This organism binds to glycolipids that contain Nglycolylneuraminic acid (Neu5Gc), in the form of Neu5Gcα2–3Galβ1–4Glc, but not to

those that contain Nacetylneuraminic acid. These two sugars differ in only a single hydroxyl group, present in the acyl substituent on the

amino group at C5 of Nglycolylneuraminic acid and absent in that of Nacetylneuraminic acid. Nglycolylneuraminic acid is found on

intestinal cells of newborn piglets, but it disappears when the animals develop and grow, and it is not formed normally by humans. This can

explain why E. coli K99 can cause often lethal diarrhea in piglets but not in adult pigs or humans.

TABLE 34.2Examples of interactions of bacterial adhesins with glycans

The relationship between microbes and the host can be quite complex. For example, colonization of germfree mice with Bacteroides

thetaiotaomicron, a normal resident microbe of the small intestine, can induce an α1–2 fucosyltransferase in the mucosal epithelial cells.

The bacteria bind to Lfucose residues and also use it as a carbon source.

TOXINS THAT BIND GLYCANSA number of secreted bacterial toxins also bind to glycans (Table 34.3). The beststudied example is the toxin from Vibrio cholera (cholera

toxin), which consists of A and B subunits in the ratio AB . Its crystal structure shows that the B subunits bind to the Galβ1–3GalNAc

moiety of GM1 ganglioside receptors through CRDs located on the base of the subunits (Figure 34.6). The A subunit is loosely held above

the plane of the B subunits, with a single αhelix penetrating through a central core created by the pentameric B subunits. Upon binding to

membrane glycolipids through the B subunits, the A subunit is delivered to the interior of the cell by an unknown mechanism. In cholera

toxin, the affinity of the B subunit for its glycan ligand (GM1) is unusually high compared to the binding of related AB toxins from

Shigella dysenteria, Bordetella pertussis, and E. coli to their glycan receptors (K in the nanomolar range for cholera toxin B subunit vs.

millimolar range for Shiga toxin B subunit binding to P determinants). In both examples, the formation of the pentameric complex greatly

5

5

dk



increases the avidity of the interaction. This phenomenon is being exploited to make oligovalent glycan ligands as protective agents against

the toxin.

TABLE 34.3Examples of receptors for bacterial toxins

FIGURE 34.6Crystal structure of cholera toxin Bsubunit pentamer with bound GM1 pentasaccharide shown from the bottom (a) andfrom the side (b). (Redrawn, with permission, from Merritt E.A., Sarfaty S., van den Akker F., L’Hoir C., Martial J.A., andHol (more...)

Bacillus thuringiensis, which lives in the soil, produces crystal toxins (Bt toxins) that can kill larval stages of plantpathogenic insects, but

they are harmless to most other organisms, including humans. Bt toxins are used in crop protection by spraying plants or by genetically

engineering crops to express the toxins. Recent work demonstrates that Bt toxins act by binding to glycolipids that line the gut in nematodes

and presumably other invertebrates (see Chapter 23). These receptors belong to the arthroseries of glycolipids and include in their structure

the characteristic ceramidelinked, mannosecontaining core tetrasaccharide GalNAcβ1–4GlcNAcβ1–3Manβ1–4GlcβCer, which is found

only in invertebrates and is conserved between nematodes and insects but is absent in vertebrates (see Chapter 24). Thus, the specificity of

Bt toxins determines their tissue and species sensitivity.

Shiga toxin produced by Shigella dysenteria and the homologous Shigalike toxins of E. coli (also called verotoxins) will bind to Galα1–

4Gal determinants on both glycolipids and glycoproteins, but only the binding to glycolipids results in cell death. The apparent preference

of most toxins for glycolipids may be related to the juxtaposition of glycolipid glycans to the membrane surface, compared to the more

distal location of glycans attached to glycoproteins and proteoglycans. Binding of a toxin or bacterium to a glycolipid also might increase

the likelihood of further interactions with the membrane (e.g., binding to another receptor or membrane intercalation).

PARASITE LECTINSIn addition to viruses and bacteria, a number of parasites also utilize glycans as receptors for adhesion (Table 34.4). Entamoeba histolytica

expresses a 260kD heterodimeric lectin that binds to terminal galactose/Nacetylgalactosamine residues on glycoproteins and glycolipids.

Binding may have a role in attachment, invasion, and cytolysis of intestinal epithelium, and it may function in binding the amoeba to

bacteria as a food source. The lectin is heterodimeric, with a transmembrane subunit of 170 kD and a glycosylphosphatidylinositol (GPI)

anchored subunit of 35 kD. The glycanbinding site is located in a cysteinerich domain. The importance of this receptor in virulence has

been established by antisense silencing of the adhesin and by expression of a dominantnegative form of the light subunit. Thus, the adhesin

is a potential target to manage E. histolytica infection (see Chapter 40).

TABLE 34.4Examples of glycan receptors for parasites

The interaction of Plasmodium falciparum (malaria) merozoites with red blood cells depends on sialic acids present on the host cell, in

particular on the major erythrocyte membrane protein glycophorin. In this organism, attachment is mediated by a family of specific sialic

acid–binding adhesin on merozoites, the most prominent of which is called EBA175 (erythrocytebinding antigen175). The adhesin shows

specificity for the type of sialic acid, with preference for Neu5Ac, rather than for 9OacetylNeu5Ac or Neu5Gc. Soluble Neu5Ac and

Neu5Acα2–6Galcontaining oligosaccharides do not competitively inhibit the binding of EBA175 to erythrocytes, but Neu5Acα2–3Gal

containing oligosaccharides are effective inhibitors, indicating that the adhesin is sensitive to the linkage of the sialic acid to the underlying

galactose. Binding to erythrocytes leads to invasion and eventual production of additional merozoites. Other organisms expressing sialic



acid adhesins can also bind to erythrocytes (e.g., influenza virus), but these interactions do not lead to productive infections in these

nonnucleated cells. Thus, under these circumstances, the erythrocyte might be considered to be a clearance mechanism for these agents (see

Chapter 3).

Plasmodiuminfected erythrocytes also express GAGbinding proteins that are thought to facilitate adherence of infected cells to tissues.

The circumsporozoite form of P. falciparum binds to heparan sulfate in a tissuespecific manner, with preferred binding to the basal surface

of hepatocytes and the basement membrane of kidney tubules. The carboxyl terminus of the protein contains positively charged residues.

Clustering of the circumsporozoite protein on the surface of the organism may generate a high concentration of positively charged residues

that facilitate binding. Recent studies show that the extent of sulfation of heparan sulfate that the parasite encounters determines whether it

migrates or productively invades host cells.

MICROBIAL GLYCAN LIGANDS FOR ANIMAL CELL LECTINSLike mammalian cells, bacteria, viruses, and parasites are covered by glycans. The enveloped viruses contain virally encoded membrane

glycoproteins and may pick up host glycoproteins and glycolipids during the budding process. Bacteria contain a cell wall composed of

peptidoglycan, teichoic acids (Grampositive organisms), and lipopolysaccharide (Gramnegative organisms) and also, in some species, a

capsule (see Chapter 20). All of these glycans can potentially interact with hostcell lectins. Thus, it is not surprising that interactions

between microbial glycans and host lectins can also aid in infection and colonization.

Trypanosoma cruzi has developed an interesting strategy of molecular camouflage in which a parasiteencoded transsialidase transfers

sialic acid from serum glycoproteins in the host to membrane proteins on its own surface. The primary function of this reaction is most

likely to cover surface glycans as a way of preventing host immune reactivity, although the transsialidase may also act as an adhesin.

Neisseria gonorrhoeae uses low levels of tissue CMPsialic acid to cover itself with sialic acid residues, making it resistant to the alternative

pathway of complement. It also appears likely that these and other sialylated pathogens interact with host via the Siglec family of sialic

acid–binding lectins (see Chapter 32). Schistosomes, which are parasitic filarial worms, contain the Lewis antigen that is also found on

human leukocytes. Because Lewis is recognized by selectins, the presence of these carbohydrates may provide a mechanism for attachment

or transcellular migration of the parasite. However, these glycans also generate a massive antiLewis antibody response in the host.

In a similar way, the capsules and lipopolysaccharide that surround bacteria, as well as yeast cell walls, contain glycans that may be

recognized by mammalian cell lectins. For example, yeast mannans are recognized by both soluble and macrophage mannosebinding

proteins, which have an important role in innate immune defense during the preimmune phase in infants. Highly virulent forms of

pathogenic bacteria such as Streptococcus contain a hyaluronan capsule, which may interact with hyaluronanbinding proteins (such as

CD44) present on hostcell surfaces and facilitate colonization. The structure and biology of these types of glycans and glycanbinding

proteins are discussed in Chapters 20, 21, and 40.

ROLE IN INFECTIOUS DISEASEAs pointed out earlier, the major function of the microbial lectins is to mediate adhesion of the organisms to host cells or tissues, which is a

prerequisite for infection to occur. This has been extensively demonstrated both in vitro (in studies with isolated cells and cell cultures) and

in vivo (in experimental animals) and is supported in some cases by clinical data. For example, lectindeficient microbial mutants often lack

the ability to initiate infection. In addition, type1 fimbrial expression is associated with the severity of urinary tract infection in children.

Moreover, mono or oligosaccharides have been shown to protect against infection by lectincarrying bacteria in experimental models.

Glycans recognized by microbial surface lectins block the adhesion of the organisms to animal cells not only in vitro, but also in vivo, and

thus protect animals against infection by such organisms. For example, coadministration of methyl αmannoside with type1fimbriated E.

coli into the urinary bladder of mice reduces significantly the rate of urinary tract infection, whereas methyl αglucoside, which is not

inhibitory to the fimbriae, has no effect. The protective effect of antiadhesive sugars has been demonstrated in a variety of studies with

different pathogenic bacteria and animals, from rabbits to monkeys. Multivalent ligands should prove even more potent.

The ability of exogenous heparin and related polysaccharides to inhibit viral replication suggests that this approach might lead to

polysaccharidebased antiviral pharmaceutical agents. As more crystal structures become available, the ability to custom design small

molecule inhibitors that fit into the carbohydraterecognition domains of adhesins should improve. Already, the structures of influenza

hemagglutinin and sialidase have suggested numerous ways to modify sialic acid to fit better into the active sites. Some of these compounds

are presently in use to limit the spread of virus (see Chapters 50 and 51).

x

x

x



FURTHER READING1. Rostand KS, Esko JD. Microbial adherence to and invasion through proteoglycans. Infect Immun. 1997;65:1–8. [PMC free article:

PMC174549] [PubMed: 8975885]

2. Kitov PI, Sadowska JM, Mulvey G, Armstrong GD, Ling H, et al. Shigalike toxins are neutralized by tailored multivalent

carbohydrate ligands. Nature. 2000;403:669–672. [PubMed: 10688205]

3. Williams SJ, Davies GJ. Protein–carbohydrate interactions: Learning lessons from nature. Trends Biotechnol. 2001;19:356–362.

[PubMed: 11513999]

4. Hooper LV, Midtvedt T, Gordon JI. How hostmicrobial interactions shape the nutrient environment of the mammalian intestine.

Annu Rev Nutr. 2002;22:283–307. [PubMed: 12055347]

5. Sharon N, Lis H. History of lectins: From hemagglutinins to biological recognition molecules. Glycobiology. 2004;14:53R–62R.

[PubMed: 15229195]

6. Spear PG. Herpes simplex virus: Receptors and ligands for cell entry. Cell Microbiol. 2004;6:401–410. [PubMed: 15056211]

7. Griffitts JS, Aroian RV. Many roads to resistance: How invertebrates adapt to Bt toxins. BioEssays. 2005;27:614–624. [PubMed:

15892110]

8. Newburg DS, RuizPalacios GM, Morrow AL. Human milk glycans protect infants against enteric pathogens. Annu Rev Nutr.

2005;25:37–58. [PubMed: 16011458]

9. Olofsson S, Bergstrom T. Glycoconjugate glycans as viral receptors. Ann Med. 2005;37:154–172. [PubMed: 16019714]

10. Sharon N. Carbohydrates as future antiadhesion drugs for infectious diseases. Biochim. Biophys. Acta. 2006;1760:527–537.

[PubMed: 16564136]

11. Sinnis P, Coppi A. A long and winding road: The Plasmodium sporozoite’s journey in the mammalian host. Parasitol Int.

2007;56:171–178. [PMC free article: PMC1995443] [PubMed: 17513164]

12. Mandlik A, Swierczynski A, Das A, TonThat H. Pili in Grampositive bacteria: Assembly, involvement in colonization and

biofilm development. Trends Microbiol. 2008;16:33–40. [PMC free article: PMC2841691] [PubMed: 18083568]

Publication Details

Author Information

Jeffrey D Esko and Nathan Sharon.

Copyright

Copyright © 2009, The Consortium of Glycobiology Editors, La Jolla, California.

Publisher

Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY)

NLM Citation

Esko JD, Sharon N. Microbial Lectins: Hemagglutinins, Adhesins, and Toxins. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd

edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 34.

Structural Evolution of Glycan Recognition by a Family of Potent HIV Antibodies

Structural evolution of glycan recognition by a family of potent HIV antibodies

Fernando Garces1,2,3,#, Devin Sok2,3,4,#, Leopold Kong1,2,3, Ryan McBride5, Helen J. Kim1, Karen F. Saye-Francisco2,3,4, Jean-Philippe Julien1,2,3, Yuanzi Hua1, Albert Cupo7, John P. Moore7, James C. Paulson5, Andrew B. Ward1,2,3, Dennis R. Burton2,3,4,8,*, and Ian A. Wilson1,2,3,6,*

1Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037, USA

2International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA

3Scripps Center for HIV/AIDS Vaccine Immunology & Immunogen Discovery, The Scripps Research Institute, La Jolla, California 92037, USA

4Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA

5Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA

6Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA

7Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA

8Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA

SUMMARY

© 2014 Elsevier Inc. All rights reserved.*co-corresponding authors (to whom correspondence should be addressed. [email protected]; [email protected]).#co-first authors

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Accession CodesCoordinates and structure factors for Fab PGT124 and its complex with gp120 and CD4 are deposited with the PDB under accession codes 4R26 and 4R2G, respectively. The Fab PGT124:SOSIP.664 trimer EM reconstruction is deposited in the EMDB under accession code EMD-2753.

SUPPLEMENTAL DATASupplemental Data includes 7 Figures and 6 Tables.

Author Contributions: Project design by F.G., D.S., A.B.W., D.R.B. and I.A.W.; X-ray work and analysis by F.G. and L.K.; EM work by H.K. and A.B.W.; glycan array work by R.M. and J.C.P.; mutational work by D.S. and K. F. S.; manuscript written by F.G, D.S., L.K., A.B.W., D.R.B. and I.A.W. All authors were asked to comment on the manuscript.This is manuscript # 26090 from The Scripps Research Institute.

NIH Public AccessAuthor ManuscriptCell. Author manuscript; available in PMC 2015 September 25.

Published in final edited form as:Cell. 2014 September 25; 159(1): 69–79. doi:10.1016/j.cell.2014.09.009.

NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

The HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield

it from recognition by the human immune system. Here we examine how a particularly potent

family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural

features to counter the glycan shield and interact with both glycan and protein components of HIV

Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a

short protein segment. Affinity maturation then leads to divergent evolutionary branches that

either focus on a single glycan and protein segment (e.g. Ab PGT124) or engage multiple glycans

(e.g. Abs PGT121-123). Furthermore, other surrounding glycans are avoided by selecting an

appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition

lessons are important for engineering proteins that can recognize or accommodate glycans.

INTRODUCTION

The HIV-1 envelope glycoprotein (Env) trimer is the sole target of the neutralizing antibody

response and the primary platform for vaccine design. However, variable loops on gp120

mediate antibody escape and extensive N-linked glycosylation shields much of the Env

protein surface from immune recognition. Additionally, many antibodies against monomeric

gp120 bind without measurable glycan involvement or show enhanced binding following

deglycosylation (Binley et al., 1998; Koch et al., 2003; Ma et al., 2011). Notwithstanding, a

number of potent broadly neutralizing antibodies (bnAbs) have recently been discovered that

bind to a heavily glycosylated region around the base of the V3 loop that we have termed a

supersite of vulnerability (Kong et al., 2013). These bnAbs include antibody families from

different germline lineages such as PGT121-123/PGT133-134/10-1074, PGT125-128/

PGT130-131 and PGT135-137 (Julien et al., 2013c; Kong et al., 2013; Mouquet et al., 2012;

Pejchal et al., 2011; Walker et al., 2011). Crystal structures of PGT128 and PGT135 in

complex with gp120 outer domain and with gp120 core, respectively, and PGT122 in

complex with the soluble, cleaved BG505 SOSIP.664 gp140 trimer (SOSIP.664) (Julien et

al., 2013a; Julien et al., 2013b; Kong et al., 2013; Pejchal et al., 2011) have enabled

molecular characterization of their glycan-dependent bnAb epitopes. Although these bnAbs

are derived from different germline lineages, they all interact with the Asn332 (N332)

glycan that is highly conserved across the majority of HIV-1 isolates. In addition, PGT128

binds the glycan at Asn301 (N301) and the base of the gp120 V3 loop, PGT135 interacts

with glycans at Asn386 (N386) and Asn392 (N392) and an extensive β-sheet motif on the

gp120 outer domain, and PGT122 contacts glycans at N301, Asn137 (N137), and Asn156

(N156), as well as protein components of the V1 and V3 loops. A family of trimer-

preferring antibodies, PG9/PG16, also recognize N156 in V1 but interact with a glycan in

V2, Asn160 (N160) at the trimer apex (Julien et al., 2013a; McLellan et al., 2011). A

common feature of these antibodies is interaction with multiple glycans and protein

components to achieve high affinity. Indeed, these same bnAbs generally have low or

undetectable affinity to single glycans (McLellan et al., 2011; Mouquet et al., 2012). For

carbohydrate binding lectins, high affinities that are relevant in vivo are achieved through

interaction with multiple glycans (Dam et al., 2000). Only one HIV-1 antibody 2G12 has

been able to attain high affinity for glycans alone by using multivalency through domain

swapping of the variable heavy chain (VH) domains, whereby two tightly linked Fabs then

bind multiple glycans in the N332 high mannose patch (Calarese et al., 2003). To achieve

Garces et al. Page 2

Cell. Author manuscript; available in PMC 2015 September 25.

NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

high affinity binding without multivalency, a combination of glycan and protein interactions

would appear to be a more general solution.

PGT122 is a member of the PGT121 family of bnAbs, which are among the most potent

antibodies identified to date. Passively administered PGT121 protects against mucosal SHIV

(chimeric simian HIV) challenge in macaques at serum concentrations achievable by

vaccination and causes a dramatic and sustained lowering of viral load in established SHIV

infection (Moldt et al., 2012; Barouch et al., 2013). The crystal structure of BG505 SOSIP.

664 with PGT122 revealed how an affinity-matured antibody from the PGT121 family

recognizes gp120 in the context of the Env trimer (Julien et al., 2013a). PGT124 is a newly

discovered bnAb from the same germline lineage as PGT121, but represents an alternative

branch in the antibody maturation process and is 89% identical in VH amino-acid sequence

to 10-1074 (Figure S1C) (Mouquet et al., 2012; Sok et al., 2014; Sok et al., 2013).

Structural comparison of representatives from these two different evolutionary branches

therefore provides an opportunity to investigate the evolution of high affinity recognition of

an epitope involving glycans and protein surfaces. Molecular details of the PGT124-gp120

interaction were determined by X-ray crystallography and EM, with additional functional

and biochemical insights from isothermal titration calorimetry (ITC), next-generation

sequencing, virus neutralization and glycan arrays. Surprisingly, PGT124 engages in a

distinct mode of interaction with the N332 supersite of vulnerability that involves a single

glycan whereas PGT122 contacts multiple glycans.

RESULTS

Structural characterization of PGT124 in complex with gp120 and CD4

To identify the PGT124 epitope, we co-crystallized PGT124 Fab in complex with a gp120

core containing a mini V3 loop (mV3) (Pejchal et al., 2011). The JRCSF gp120 mV3 core

was produced in a GnTI−/− mutant cell line that yields Man5-9GlcNAc2 oligomannose

glycans (Chang et al., 2007; Reeves et al., 2002). The complex was deglycosylated by

Endoglycosidase H (EndoH) to remove glycans not protected by PGT124 binding and two-

domain soluble CD4 was added to facilitate crystallization. The crystal structure was

determined at 3.3 Å with four ternary complexes in the asymmetric unit (Table S1 and S6).

The most prominent interaction of PGT124 with gp120 is with a single Man8GlcNAc2

glycan (Man8) linked to N332 and a small protein segment at the V3 loop base (Figure 1A,

B). Structurally, both protein and glycan components make comparable contributions to the

PGT124 epitope, as the bnAb buries 428 Å2 of gp120 polypeptide and 480 Å2 of N332

Man8 glycan (Figure 1C). While the N332 glycan was protected from EndoH

deglycosylation by the antibody prior to crystallization, most other glycans appear to be

cleaved, as indicated by electron density for only single N-acetylglucosamine (GlcNAc)

moieties at N262, N276, N295, N301, N339, N355, N386, N392, N411 and N448. Thus, it

is unlikely that the N301 glycan near the base of V3 makes productive contacts with

PGT124. However, additional electron density at N295 and N339 could be assigned to two

core GlcNAcs (data not shown) in one of the four copies in the asymmetric unit, suggesting

partial protection from cleavage by EndoH. Electron density for gp120 was mostly



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

unambiguous except for the mV3 tip, which is disordered. However, the V3 base is highly

ordered as PGT124 makes its only contacts with the gp120 polypeptide in this region

(Figure 2A). Superposition of the unliganded PGT124 structure at 2.5 Å (Table S1) with its

bound form gave an all-atom root mean square deviation (RMSD) of 0.6 Å, indicating no

major changes in the antibody on gp120 binding (Figure S1A).

To characterize the PGT124 interaction in the context of the SOSIP.664 Env trimer

(Depetris et al., 2012; Sanders et al., 2002), we derived negative-stain electron microscopy

(EM) images at 17.8 Å resolution. Three Fabs are bound per trimer with each oriented

diagonally relative to the trimer axis (Figure S2A). The PGT122:SOSIP.664 crystal structure

(PDB ID:4NCO) fits extremely well into the PGT124:SOSIP.664 EM reconstruction,

indicating both bnAbs approach Env at a similar angle (Figure 1D).

Molecular basis of PGT124 recognition of gp120 polypeptide

PGT124 recognizes the 324GDIR327 region at the V3 loop base. As other N332-dependent

antibodies, including PGT128, bind this same sequence, this motif may be a common anchor

point. For PGT124, Ser30 and Ser93 in CDR L1 and L3, respectively, interact with the gp120

Asp325 carboxylate (Figure 2A), which is also involved in stacking interactions with CDR

H3 Tyr100B. Remarkably, PGT124 utilizes three different CDR loops to target Asp325.

Furthermore, a salt bridge is formed between CDR H3 Glu100I and gp120 Arg327. However,

these side-chain mediated PGT124 interactions contrast with the backbone-mediated

recognition of the same 324GDIR327 region by PGT128, thus highlighting substantial

differences in how antibodies from different lineages recognize the same region in an

epitope (Figure 2B). Furthermore, Asp325 can be substituted by Asn without affecting

PGT124 interaction as HIV-1 isolates such as CNE20 and CAAN5342.A2 with 324GNIR327

can be neutralized. This 325D/NxR327 motif is 94.1% conserved across HIV-1 strains, as

calculated using 3,045 aligned gp120 sequences (Los Alamos HIV Database). As expected,

mutation of GDIR to GAIA completely abrogated PGT124 binding to gp120 in an ITC

assay (Figure S3A, B). A JRCSF pseudovirus variant containing GAIA was poorly

neutralized by PGT124 compared to wild type, while the same mutations had no effect on

neutralization by PGT128 (Figure 2D). Thus, PGT124 interacts with the highly conserved

V3 base in a distinct manner from PGT128. Accordingly, successful elicitation of these two

types of bnAbs may require differently designed immunogens.

Further comparison between the PGT124:gp120 core-mV3 and PGT128:ODmV3 (PDBID:

3TYG) structures confirmed the GDIR motif is structurally similar (1.0 Å Cα RMSD)

although significant flexibility is observed in the overall V3 loop conformation (2.4 Å Cα

RMSD) where the observed ends of the V3 loop shift by 10 Å and 5 Å (Figure 2C). The

PGT128 V3 conformation is likely affected by antibody binding as extensive interactions

are also made with the N301 glycan within the mV3.

The PGT124:core-mV3 crystal structure was also compared with the PGT122:SOSIP.664

trimer structure (PDB ID:4NCO), where superposition indicates that the V3 base, and the

GDIR motif in particular, adopts similar but not identical, conformations (Figure S4),

suggesting binding does not substantially perturb the gp120 conformation in monomer or

trimer context.



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Man8/9 linked to Asn332 is the primary PGT124 glycan contact in the N332 supersite of vulnerability

The PGT124 structure reveals the molecular basis for the highly focused N332 glycan

interaction. The antibody engages carbohydrate primarily at the junction of the light (LC)

and heavy (HC) chains (Figure 3A) and makes multiple interactions to gp120 via CDR L2,

light chain framework 3 (FR L3) and the 24-residue CDR H3 that forms an extended β-

hairpin (Figure 3A). The long H3 loop penetrates the glycan shield to reach the gp120

protein surface and its extended backbone packs along the entire length of the N332 glycan.

Additional glycan interactions are made with a pocket formed by CDR L2, FR L3 and CDR

H3, where the C-mannose moiety forms hydrogen bonds and van der Waals interactions

(Figure 3D). Site-directed alanine mutagenesis confirms that AsnL51 and ArgH100, which

directly contact the glycan, moderately affect PGT124 neutralization, whereas IleL66b has a

weaker effect (Figure S5). Together, these extensive glycan contacts, in combination with

interactions with the GDIR motif, appear to be sufficient to attain high affinity binding,

without engaging other glycans, as with PGT128 and PGT135. Nevertheless, although

PGT124 contacts the three mannose moieties on the D1 arm, only two seem to impact

neutralization (Figure S5). Moreover, PGT124 can neutralize viruses grown in different cell

lines or in the presence of glycosidase inhibitors (Figure 3E), presumably because the N332

glycan composition is not significantly impacted as it is protected from modifying enzymes

by the dense oligomannose patch on the gp120 outer domain (Bonomelli et al., 2011).

Co-crystal structures of gp120 with a variety of N332-dependent bnAbs (PGT128 (PDB ID:

3TYG), PGT135 (PDB ID:4JM2), and PGT124) allow assessment of the structure and

conformational flexibility of the N332 glycan. First, superimposition of their gp120 protein

components shows that the D1, D2 and D3 arms of the N332 glycan generally project in the

same direction. However, while the PGT135- and PG128-bound N332 glycans varying by

only 8° in an arc from each other, the PGT124-bound N332 deviates by 20-25° from its

corresponding orientations in the PGT128 and PGT135 complexes, bending closer towards

the V3 loop (Figure 3A-C and Figure S6A, B). Otherwise, the N332 glycan seems to have a

rather rigid architecture. Superimposition only on the N332 glycan reveals remarkably good

correspondence of the glycan conformation with only slight deviations (Figure S6C) as

reflected in all-atom RMSD’s of 2.5 Å and 2.9 Å for N332-PGT124 vs N332-PGT135 and

N332-PGT124 vs N332-PGT128 structures, respectively. Thus, the N332 glycan has a

consistent similar general orientation, but antibodies can fix some of the apparently limited

conformational heterogeneity. Finally, PGT124 binds a different face of the glycan than

PGT135, which interacts with the N332 D2 and D3 arms rather than D1. In contrast,

PGT124 contacts the same glycan face as PGT128, which binds the D1 and D3 arms.

Consequently, this variation in N332 glycan recognition and binding modes creates specific

footprints for each antibody.

Although derived from the same putative germline precursor, PGT124 and PGT122 make

different sets of glycan contacts. PGT124 contacts only N332 on the JRCSF gp120 core,

while PGT122 contacts four different glycans on the BG505 SOSIP.664 trimer (Julien et al.,

2013a). Indeed, in the absence of the N332 glycan, PGT122 requires N137 and N301 to

neutralize the BG505 virus (Julien et al., 2013a). To determine if PGT124 also depends on



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

these glycans, we eliminated single glycan sites in the V1/V2 loops and in the high-mannose

patch of Env; these changes had little to no effect on PGT124 neutralization (Table S2).

Consistent with the structural data, only N332 glycan removal abrogated PGT124

neutralization across a number of isolates (Sok et al., 2014). In contrast, PGT121 exhibits

isolate-specific effects in the presence or absence of N332 (Table S2) (Sok et al., 2013),

likely reflecting its ability to utilize alternative glycans at either N137 or N301 (Sok et al.,

2014). Removal of these glycan sites individually did not affect or even enhance PGT121

activity, implying that each glycan interaction is relatively weak. Thus, it appears that the

glycans are recognized in combination with the protein component via a cooperative

interaction to achieve a high-affinity interaction. Although PGT124 and PGT121/PGT122

derive from the same germline genes and have high structural homology, they have diverged

significantly in how they interact with their epitopes centered on the N332 glycan.

Nevertheless, the entire constellation of glycans likely dictates the approach angle of these

antibodies to the Env trimer where either several (PGT122) or one (PGT124) glycans are

contacted and others are avoided.

We previously reported that removal of the N332 glycan abrogates PGT124 neutralization

for the overwhelming majority of isolates (Sok et al., 2014). To determine how PGT124

neutralizes HIV in the absence of N332, we investigated a small set of representative

isolates (Table S3). Neighboring glycan sites were removed in combination with N332 by

alanine mutagenesis, and the double mutants were tested to see whether PGT124 was

dependent on them for neutralization. Loss of neutralization was found for removal of N295,

N301, N339, and N392 glycan sites in combination with N332 removal, but little to no

effect was seen with single mutations (Table S3). Accordingly, for a small subset of viruses,

PGT124 is capable of utilizing nearby glycans when N332 is not present (Table S3).

PGT124 binding does not appear to be dependent on other surrounding glycans on the HIV-1 trimer

PGT124 primarily contacts N332 whereas PGT122 also contacts N137, N156, and N301

(Figure 1) (Julien et al., 2013a). Therefore, we analyzed sequences in this family in the

context of known structures to assess the evolution of their binding sites during antibody

maturation. Residues in common in PGT122 and PGT124 CDRs mediate contacts to N332

and GDIR (Figure 4). Indeed, almost all of these contact residues are also present in the

germline (GL) and retained in various intermediates (3H, 32H, 9H for the heavy chain and

3L for the light chain) as well as in the affinity-matured antibodies from different branches

of the phylogenetic tree (PGT121-124). Thus, N332 glycan and GDIR recognition appears

to be the key event in initiating the antibody response.

Comparison of HIV neutralization by inferred intermediates and affinity-matured antibodies

against various glycan mutants reveals considerable complexity in glycan epitope

recognition. We therefore focused on the four glycan sites (N137, N156, N301, and N332)

that mediate binding of PGT122 to the BG505 isolate. We tested neutralization of antibody

3H+3L, the least-mutated inferred intermediate for this lineage, as well as 32H+3L that is an

inferred intermediate of PGT124, and 9H+3L as an inferred intermediate of PGT121-123.

First, we observed loss of neutralization for 3H+3L upon removal of individual glycan sites



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

at N301 and N332, as previously reported (Sok et al., 2013), implying use of both glycan

sites early in this lineage (Table S4). Neutralization was reduced after N301 removal for

32H+3L and 9H+3L with complete loss after N332 removal, suggesting a somewhat

reduced dependence on N301 during affinity maturation relative to N332. A similar pattern

was observed for other isolates (Sok et al., 2013). Strikingly, N137 removal resulted in

enhanced neutralization for both inferred intermediate antibodies and for the affinity-

matured antibodies (Table S4), suggesting the N137 glycan plays a largely obstructive role.

However, in the absence of the N332 glycan, it appears that the N137 glycan can play a

positive binding role. If N332 and N137 are both eliminated, antibodies from the PGT121

family fail to neutralize virus. The overall conclusion is that the N332 glycan is

preferentially used by all antibodies of the PGT121 lineage but the N137 glycan can be

utilized by PGT121-123 if N332 is absent (Table S5). Moreover, this effect is also observed

to a lesser degree for N301 and N156 glycans that do not generally contribute to binding in

the presence of N332, but become important for PGT121-123 in the absence of N332 (Table

S4). In contrast, for the PGT124 branch, the N332 glycan appears to be sufficient for

neutralization and contact with N137 is avoided (Table S4 and S5). Thus, markedly different

epitope recognition is observed for antibodies derived from the same germline lineage. With

regard to the N301 glycan, PGT124 CDR H2 is shifted away from its equivalent position in

PGT122 (Figure 5C) and towards N301 glycan (Figure 5D). In fact, PGT124 would clash

with N301 if it were to maintain the same orientation it adopts in the PGT122 complex.

Thus, PGT124 appears to avoid this glycan consistent with apparent cleavage by EndoH in

the presence of PGT124 and no visible electron density.

As previously observed, glycan arrays indicated that PGT121 binds bi-antennary complex

glycans containing terminal sialic acid moieties with α-6 linkages (Julien et al., 2013c;

Mouquet et al., 2012), but not high-mannose glycans (Figure 6 A-C). PGT128 and 2G12

only bind high-mannose glycans on glycan arrays (Calarese et al., 2003; Pejchal et al.,

2011). In contrast, PGT124 does not bind any glycans on these arrays (Figure 6 A-C). The

lack of complex glycan binding is consistent with the structural evidence that PGT124 does

not interact with V1/V2 glycans, which are likely complex. Indeed, similar data were

reported for 10-1074, a closely related somatic variant of PGT124 (Figure S1B) (Mouquet et

al., 2012). However, lack of detectable high-mannose binding to PGT124 on the arrays is

more puzzling but consistent with weak interaction with the N332 glycan that requires

cooperative interaction with the GDIR motif to attain high affinity binding. For WEAU, one

of the few HIV-1 strains in which N332A mutation does not abrogate PGT124

neutralization, virus produced in the presence of kifunensine, which results in only

Man8-9GlcNAc2 glycoforms, is more sensitive to PGT124 neutralization than wild-type

virus, suggesting complex glycans are not required for PGT124 neutralization even in the

absence of N332, in contrast to PGT121 (Figure S7).

PGT121-123 allosterically inhibit CD4 binding to gp120 by interacting with the V1/V2 loop

(Julien et al., 2013c). To test whether this mechanism also applies to PGT124, we performed

sequential ITC binding experiments in which CD4 was titrated into a solution containing

full-length gp120 pre-complexed with PGT124 or vice-versa. We found no difference in

PGT124 or CD4 binding whether or not the other was precomplexed with full-length gp120



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

(PGT124: Kd of 38nM vs 34nM precomplexed with CD4; CD4: Kd of 55nM vs 50nM

precomplexed with PGT124). Thus, PGT124 does not inhibit gp120-CD4 binding, either

allosterically or otherwise, which further highlights differences in epitope recognition

compared to PGT121 (Figure S3E).

Discussion

Following HIV-1 infection, a proportion of individuals develop potent broadly neutralizing

antibody responses over time. Typically, sets of monoclonal antibodies can be isolated that

consist of somatic variants arising from a single germline precursor but individual variants

can differ considerably in sequence, neutralization profiles and breadth. Investigation of

these somatic variants can aid in understanding what to expect from a vaccine-induced

polyclonal antibody response to a given epitope and highlight features that might be

gainfully incorporated into a vaccine immunogen and in vaccination strategies designed to

elicit antibodies to that epitope.

To this end, we performed a structural study of members of the highly potent PGT124

family of bnAbs. PGT121, for example, protects against virus challenge at exceptionally low

serum titers in macaque models (Moldt et al., 2012). Previous work also noted differences in

glycan site recognition between somatic variants (Sok et al., 2014) and phylogenetic studies

reported divergence in affinity maturation between PGT121-123 and 10-1074/PGT124

(Mouquet et al., 2012; Sok et al., 2013). We therefore investigated these phenotypic

differences by comparing epitope recognition between somatic variants PGT122 and

PGT124 in atomic detail.

The overall binding site architectures of the PGT124/10-1074, PGT121-123, and germline

precursor antibodies are strikingly similar; all contain a closed face (Julien et al., 2013c;

Mouquet et al., 2012) on one side of the paratope for binding the N332 glycan, and similarly

oriented long CDR loops that reach through to the protein surface below (Figure S1B). All

antibodies also have an equivalent open face with a U-shaped depression; for PGT122, this

face contacts surrounding glycans, whereas for PGT124, it enables avoidance of neighboring

glycans. These observations suggest that, from the initial recombination event, the overall

shape of the germline precursor is compatible with penetration of the glycan shield on the

Env trimer in the region of N332 (Figure S1B). Indeed, the germline precursor already

contains 8 of the 11 residues that are used by PGT124 to recognize the N332 glycan (5

residues) and the GDIR peptide sequence (6 residues) (Figure 4). A three amino-acid

insertion in FR L3, which occurred early in the maturation process, makes key contacts to

the N332 glycan, highlighting its importance for the binding and neutralization by this

antibody family (Sok et al., 2013),

However, the structural analyses, as well as neutralization and binding assays, revealed a

remarkable divergence in glycan epitope recognition by this antibody lineage that could

have been driven by changes in the glycan sites over the course of chronic infection,

although this hypothesis cannot be tested directly as longitudinal samples are not available.

However, enhanced neutralization by all members of this lineage in the absence of the N137

glycan suggests that the initial encounter with this glycan was unfavorable, if it was present,

but likely contributed to selecting the overall antibody shape required to bind the N332



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

glycan, penetrate through the glycan shield to access the V3 base, and avoid unfavorable

contacts with other neighboring glycans. Following this initial event, antibody maturation

appears to have proceeded along two different paths. Via the PGT124 route, affinity

maturation appears to have focused on recognition on the V3 base and the N332 glycan. For

PGT121-123, affinity maturation appears to have expanded the recognition to up to four

glycans, including N137, depending upon isolate context, which likely could counter

potential virus escape through deletion of the N332 glycan (Figure 7). Even though the

PGT124 epitope is substantially smaller than PGT121-123, PGT124’s potency and breadth

match the other antibodies (Sok et al., 2014). One possible explanation is that interactions

with the protein component substitute for binding to multiple glycans. Accordingly, the

GDIR motif is just as important as the N332 glycan for binding and neutralization for most

isolates (Figures 2D, 3E and Figure S3B-C). Thus, the single glycan at N332 is necessary

but not sufficient since the GDIR motif is also required for PGT124 binding and

neutralization.

Our observations also highlight the inherent flexibility in how glycans can serve as binding

sites for antibodies as well as for other proteins. Furthermore, N137 glycan deletion in the

presence of the N332 glycan enhances neutralization of BG505, indicating that this glycan

can restrict antibody recognition. In contrast, deletion of both N137 and N332 totally

abrogates PGT121-123 neutralization against the BG505 strain (Figure 7 and Table S4).

Thus, the same glycan can participate in or shield a neutralizing epitope. For example,

deletion of the N156 glycan has no effect on neutralization by any PGT121 lineage antibody

except when N332 is absent, where some dependency is now observed (PGT122 and

PGT123) (Table S4). In contrast, N332 makes productive contacts with all antibodies,

confirming its role in the epitope core. The N301 glycan is important for neutralizing

activities of the 3H precursor and, to a lesser extent, 9H and 32H precursors (Table S4). In

contrast, in the absence of N332, PGT121 and particularly PGT122 show some dependency

on N301 while deletion of both glycans completely abrogates binding of PGT123 (Table

S4). In summary, several glycans may be involved in initiating an early PGT121 family

response but different maturation pathways result in divergent glycan recognition profiles.

This complexity in glycan recognition may be generalized to other antibodies such as PG9

and PG16 (Pancera et al., 2013; Walker et al., 2009), which both recognize N156 and N160

in V1/V2, but PG9 has evolved a preference for N160 while PG16 favors N156 (McLellan

et al., 2011; Pancera et al., 2013).

These results also have implications for protein-glycan recognition and vaccine design.

Broader recognition of glycoproteins or glycopeptides can be achieved by focusing

recognition on the conserved core of otherwise variable glycans. Secondly, because of such

conserved features, neighboring glycans may be able to substitute for some variation in

glycan location. Thirdly, affinity can be enhanced by binding to multiple glycans even

within a single binding site and also by latching on to the conserved regions of the protein,

albeit even very short segments. For vaccine design, further considerations must be taken

into account. For the PGT121 family, immunogens would first be designed to select for

germline antibodies with relatively long CDR H3 lengths (26 amino acids) that could insert

between the glycans and reach the underlying protein surface below. An appropriate

antibody shape would be required for intimate binding of the N332 glycan (closed face in



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

the PGT121 family), and another cavity (open face) to contact (PGT121-123) or avoid

(PGT124) surrounding glycans. To facilitate initial engagement, an immunogen without the

N137 glycan may enhance epitope recognition as illustrated for engagement of germline

antibodies to the CD4 binding site by removal of a blocking glycan (Jardine et al., 2013). It

is not yet clear whether removal of a blocking glycan would compromise selection of an

appropriately shaped antibody. Following this initial selection event, modified immunogens

would be used to drive clonal diversity of the selected antibody germline family that could

include adding back or altering glycans to emulate events that likely occurs during virus

evolution under immune pressure. To achieve a productive angle of approach of the

antibody in the context of the Env trimer, the immunogen would likely require a native

glycan shield or native-like Env structure to encourage accommodation of potentially

obstructive neighboring glycans and drive somatic mutations to productively utilize

neighboring glycans. Thus, these results provide new insights into protein recognition of

glycosylated proteins and demonstrate the remarkable ability of the human immune system

to find and evolve multiple solutions for high affinity antibody binding of complex epitopes

so as to neutralize highly variable and highly glycosylated viruses, such as HIV.

EXPERIMENTAL PROCEDURES

Expression and purification of proteins

CD4, was expressed in E. coli BL-21 DE3* cells in LB media and purified by affinity

chromatography followed by size exclusion chromatography (SEC). PGT124 Fab was

produced in FreeStyle™ 293F cells (Invitrogen) and purified by affinity chromatography

followed by cation exchange chromatography and SEC. JRCSF gp120 core was produced in

FreeStyle™ 293S. Protein was purified using a GN Lectin column, followed by SEC.

BG505 SOSIP.664 trimer was expressed in FreeStyle™ 293F or 293S cells and purified

using a 2G12-coupled affinity matrix followed by SEC (Supplemental Experimental

Procedures).

Formation of protein complexes, crystallization and data collection

Complexes were formed by combining gp120:ligands in a 1:1.2 molar ratio, followed by

deglycosylation with Endoglycosidase H before being purified by SEC. Crystals of

PGT124-gp120coremV3-CD4 ternary complex were obtained from 2.4 M (NH4)2SO4, 0.1

M Tris pH 8.0 and 13% glycerol that diffracted to 3.3Å resolution (Table S1). Unliganded

PGT124 Fab crystals appeared from 20% (w/v) PEG 4000, 0.2M MgCl2, 0.1 M Tris-HCl

pH 8.5 and diffracted to 2.5 Å (Supplemental Experimental Procedures and Table S1).

Structure determination and refinement

The unliganded PGT124 structure was solved by molecular replacement using Phaser with

the PGT122 Fab structure (PDB 4JY5) as the initial model, in which the CDR loops had

been deleted. For the ternary complex, multiple components were used for phasing: CD4-

gp120 (PDB ID:4JM2) and the high-resolution unliganded PGT124 Fab. Model building

and refinement were carried out using Coot (Emsley and Cowtan, 2004) and Phenix (Adams

et al., 2010), respectively. Table S6 shows the components that were included in the final

model. Rcryst and Rfree for the unliganded structure are 23.5% and 26.7%, and 21.0% and



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

26.3% for the complex (Table S1). Buried molecular surface areas were analyzed with the

Molecular Surface Package (Connolly, 1993) using a 1.7 Å probe radius. The Fab residues

were numbered according to Kabat (Martin, 1996) and gp120 is numbered following the

Hxbc scheme (Ratner et al., 1987).

Isothermal titration calorimetry

ITC binding experiments were performed using a MicroCal Auto-iTC200 instrument (GE).

All proteins were extensively dialyzed against a buffer containing 20 mM Tris, 150 mM

NaCl, pH 7.4 and protein concentrations were adjusted and confirmed using calculated

extinction coefficients and absorbance at 280 nm. In the syringe, ligand was either PGT124

Fab or sCD4 at concentrations ranging between 60-125 μM. The gp120 monomer was in the

cell at concentrations ranging between 3.5-10 μM. Two-protein binding experiments were

performed as follows: cell at 25°C, 16 injections of 2.5 μl each, injection interval of 180 s,

injection duration of 5 s, and reference power of 5 μcals. To perform sequential binding

experiments, the mixed sample from the first titration was left in the cell and the

concentration of the HIV-1 component was recalculated based on the dilution from the first

experiment (approximately ~88% of the initial concentration). Subsequently, either PGT124

Fab or sCD4 was added in a second titration. To calculate the affinity constants (KD), the

molar reaction enthalpy (ΔH) and the stoichiometry of binding (N), Origin 7.0 software was

used by fitting the integrated titration peaks using a single-site binding model.

Antibody and envelope substitutions

Substitutions in the PGT124 paratope, the HIV-1 Env glycoprotein, and the JRCSFmV3

construct were introduced using QuikChange site-directed mutagenesis (Stratagene, La

Jolla, CA). Substitutions were verified by DNA sequencing (Retrogen, San Diego, CA).

Neutralization assays

Neutralization activity of antibodies against pseudovirus in TZM-bl cells was determined as

described previously (Li et al., 2005).

Electron microscopy (EM)

PGT124 Fab in complex with BG505 SOSIP.664 gp140 produced in HEK 293S cells were

analyzed by negative stain EM. A 3 μL aliquot of 10 μg/ml of the complex was applied for

15s onto a glow discharged, carbon coated 400 Cu mesh grid and stained with 2% uranyl

formate for 20s. Grids were imaged using a FEI Tecnai T12 electron microscope operating

at 120 kV using 52,000 × magnification and electron dose of 25 e−/Å2, resulting in a pixel

size of 2.05 Å at the specimen plane. Images were acquired with a Tietz 4k × 4k CCD

camera in 5° tilt increments from 0° to 55° at a defocus of 1000 nm using LEGINON

(Suloway et al., 2005).

Image processing

Particles were picked automatically by using DoG Picker and put into a particle stack using

the Appion software package (Lander et al., 2009; Voss et al., 2009). Initial reference-free

2D class averages were calculated using unbinned particles via the Xmipp Clustering 2D



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Alignment and sorted into 400 classes (Sorzano et al., 2010). Particles corresponding to the

complexes were selected into a substack and another round of reference-free alignment was

carried out using Xmipp Clustering 2D alignment and IMAGIC softwares (van Heel et al.,

1996). To generate an ab initio 3D starting model, a template stack of 120 images of 2D

class averages was used with imposing C3 symmetry. This starting model was refined

against 12,245 raw particles for 30 cycles using EMAN (Ludtke et al., 1999). The resolution

of the final reconstruction was calculated to be 17.8 Å using an FSC cut-off of 0.5 (Figure

S2).

Glycan array production

Glycan arrays were custom printed on a MicroGridII (Digilab) contact microarray robot

equipped with StealthSMP4B microarray pins (Telechem) as previously described. Briefly,

samples of each glycan were diluted to 100uM in 150 NaPO4 buffer, pH 8.4. Aliquots of

10uL were loaded in 384-well plates and imprinted onto NHS-activated glass slides (SlideH,

Schott/Nexterion) with 7 arrays, each containing 6 replicates of each sample at both

concentrations. Printed slides were humidified post-print for 1h and desiccated overnight.

Remaining NHS-ester residues were quenched by immersing slides in 50mM Ethanolamine

in 50mM borate buffer, pH 9.2, for 1h. Blocked slides were washed with water, spun dry

and stored at room temperature until use.

Glycan array screening and analyses

Antibodies were diluted to 30ug/mL + 15ug/mL anti-human-IgG-RPE (JacksonImmuno) in

PBS + 0.05% Tween-20. The prepared mixture was incubated for 30 min on ice and

incubated on the array surface in a humidified chamber for 1 hour. Slides were subsequently

washed by successive rinses with PBS-T, PBS and deionized H2O. Washed arrays were

dried by centrifugation and immediately scanned for FITC and R-PE signal on a Perkin-

Elmer ProScanArray Express confocal microarray scanner. Fluorescent signal intensity was

measured using Imagene (Biodiscovery) and mean intensity minus mean background was

calculated and graphed using MS Excel.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgements

We are very grateful to N. Laursen and X. Dai for assistance with data processing; A. Irimia, and A. Sarkar for assistance with glycan refinement; N. Laursen and R. Stanfield for helpful discussions; M. Elsliger for computer support; M. Deller and H. Tien for crystallization screening; C. Corbaci for help with preparing figures; and J.P. Verenini for manuscript formatting. X-ray data sets were collected at the Stanford Synchrotron Radiation Lightsource (SSRL), a Directorate of the SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research; NIH’s National Center for Research Resources, Biomedical Technology Program (P41RR001209); and the National Institute of General Medical Sciences (NIGMS). Electron microscopy data were collected at the National Resource for Automated Molecular Microscopy (NRAMM) at the Scripps Research Institute, which is supported by US National Institutes of Health (NIH) through the National Center for Research Resources’ P41 program (RR017573). This work was supported by the International AIDS Vaccine Initiative Neutralizing Antibody Center; by the Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID UM1 AI00663) (A.B.W., D.R.B., I.A.W.), by the HIV Vaccine Research and Design (HIVRAD) program (P01 AI832362 and R37



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

AI36082) (J.P.M., A.B.W., I.A.W.), by NIH RO1 GM046192 (I.A.W.), by NIH R01 AI332932 (DRB) and by the Joint Center of Structural Genomics (JCSG) funded by the NIH NIGMS, Protein Structure Initiative (U54 GM094586) (I.A.W.).

References

Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 2010; 66:213–221. [PubMed: 20124702]

Barouch DH, Whitney JB, Moldt B, Klein F, Oliveira TY, Liu J, Stephenson KE, Chang HW, Shekhar K, Gupta S, et al. Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature. 2013; 503:224–228. [PubMed: 24172905]

Bennett A, Liu J, Van Ryk D, Bliss D, Arthos J, Henderson RM, Subramaniam S. Cryoelectron tomographic analysis of an HIV-neutralizing protein and its complex with native viral gp120. J. Biol. Chem. 2007; 282:27754–27759. [PubMed: 17599917]

Binley JM, Wyatt R, Desjardins E, Kwong PD, Hendrickson W, Moore JP, Sodroski J. Analysis of the interaction of antibodies with a conserved enzymatically deglycosylated core of the HIV type 1 envelope glycoprotein 120. AIDS Res. Hum. Retroviruses. 1998; 14:191–198. [PubMed: 9491908]

Bonomelli C, Doores KJ, Dunlop DC, Thaney V, Dwek RA, Burton DR, Crispin M, Scanlan CN. The glycan shield of HIV is predominantly oligomannose independently of production system or viral clade. PLoS One. 2011; 6:e23521. [PubMed: 21858152]

Calarese DA, Scanlan CN, Zwick MB, Deechongkit S, Mimura Y, Kunert R, Zhu P, Wormald MR, Stanfield RL, Roux KH, et al. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science. 2003; 300:2065–2071. [PubMed: 12829775]

Chang VT, Crispin M, Aricescu AR, Harvey DJ, Nettleship JE, Fennelly JA, Yu C, Boles KS, Evans EJ, Stuart DI, et al. Glycoprotein structural genomics: Solving the glycosylation problem. Structure. 2007; 15:267–273. [PubMed: 17355862]

Connolly ML. The molecular surface package. J. Mol. Graph. 1993; 11:139–141. [PubMed: 8347567]

Dam TK, Roy R, Das SK, Oscarson S, Brewer CF. Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the “multivalency effect”. J. Biol. Chem. 2000; 275:14223–14230. [PubMed: 10799500]

Depetris RS, Julien JP, Khayat R, Lee JH, Pejchal R, Katpally U, Cocco N, Kachare M, Massi E, David KB, et al. Partial enzymatic deglycosylation preserves the structure of cleaved recombinant HIV-1 envelope glycoprotein trimers. J. Biol. Chem. 2012; 287:24239–24254. [PubMed: 22645128]

Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2004; 60:2126–2132. [PubMed: 15572765]

Jardine J, Julien JP, Menis S, Ota T, Kalyuzhniy O, McGuire A, Sok D, Huang PS, MacPherson S, Jones M, et al. Rational HIV immunogen design to target specific germline B cell receptors. Science. 2013; 340:711–716. [PubMed: 23539181]

Julien JP, Cupo A, Sok D, Stanfield RL, Lyumkis D, Deller MC, Klasse PJ, Burton DR, Sanders RW, Moore JP, et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science. 2013a; 342:1477–1483. [PubMed: 24179159]

Julien JP, Lee JH, Cupo A, Murin CD, Hoffenberg S, Caulfield MJ, King CR, Marozsan A, Klasse PJ, Sanders RW, et al. Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9. Proc. Natl. Acad. Sci. USA. 2013b; 110:4351–4356. [PubMed: 23426631]

Julien JP, Sok D, Khayat R, Lee JH, Doores KJ, Walker LM, Ramos A, Diwanji DC, Pejchal R, Cupo A, et al. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. PLoS Pathog. 2013c; 9:e1003342. [PubMed: 23658524]

Kabsch W. Xds. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2010; 66:125–132. [PubMed: 20124692]

Koch M, Pancera M, Kwong PD, Kolchinsky P, Grundner C, Wang L, Hendrickson WA, Sodroski J, Wyatt R. Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition. Virology. 2003; 313:387–400. [PubMed: 12954207]



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Kong L, Lee JH, Doores KJ, Murin CD, Julien JP, McBride R, Liu Y, Marozsan A, Cupo A, Klasse PJ, et al. Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120. Nat. Struct. Mol. Biol. 2013:796–803. [PubMed: 23708606]

Lander GC, Stagg SM, Voss NR, Cheng A, Fellmann D, Pulokas J, Yoshioka C, Irving C, Mulder A, Lau PW, et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 2009; 166:95–102. [PubMed: 19263523]

Li M, Gao F, Mascola JR, Stamatatos L, Polonis VR, Koutsoukos M, Voss G, Goepfert P, Gilbert P, Greene KM, et al. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 2005; 79:10108–10125. [PubMed: 16051804]

Ludtke SJ, Baldwin PR, Chiu W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 1999; 128:82–97. [PubMed: 10600563]

Ma BJ, Alam SM, Go EP, Lu X, Desaire H, Tomaras GD, Bowman C, Sutherland LL, Scearce RM, Santra S, et al. Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies. PLoS Pathog. 2011; 7:e1002200. [PubMed: 21909262]

Martin AC. Accessing the Kabat antibody sequence database by computer. Proteins. 1996; 25:130–133. [PubMed: 8727325]

McLellan JS, Pancera M, Carrico C, Gorman J, Julien JP, Khayat R, Louder R, Pejchal R, Sastry M, Dai K, et al. Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature. 2011; 480:336–343. [PubMed: 22113616]

Moldt B, Rakasz EG, Schultz N, Chan-Hui PY, Swiderek K, Weisgrau KL, Piaskowski SM, Bergman Z, Watkins DI, Poignard P, Burton DR. Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo. Proc. Natl. Acad. Sci. USA. 2012; 109:18921–18925. [PubMed: 23100539]

Mouquet H, Scharf L, Euler Z, Liu Y, Eden C, Scheid JF, Halper-Stromberg A, Gnanapragasam PN, Spencer DI, Seaman MS, et al. Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies. Proc. Natl. Acad. Sci. USA. 2012; 109:E3268–3277. [PubMed: 23115339]

Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Meth. Enzymol. 1997; 276:307–326.

Pancera M, Shahzad-Ul-Hussan S, Doria-Rose NA, McLellan JS, Bailer RT, Dai K, Loesgen S, Louder MK, Staupe RP, Yang Y, et al. Structural basis for diverse N-glycan recognition by HIV-1-neutralizing V1-V2-directed antibody PG16. Nat. Struct. Mol. Biol. 2013; 20:804–813. [PubMed: 23708607]

Pejchal R, Doores KJ, Walker LM, Khayat R, Huang PS, Wang SK, Stanfield RL, Julien JP, Ramos A, Crispin M, et al. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science. 2011; 334:1097–1103. [PubMed: 21998254]

Ratner L, Fisher A, Jagodzinski LL, Mitsuya H, Liou RS, Gallo RC, Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res. Hum. Retroviruses. 1987; 3:57–69. [PubMed: 3040055]

Reeves PJ, Callewaert N, Contreras R, Khorana HG. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA. 2002; 99:13419–13424. [PubMed: 12370423]

Sanders RW, Vesanen M, Schuelke N, Master A, Schiffner L, Kalyanaraman R, Paluch M, Berkhout B, Maddon PJ, Olson WC, et al. Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J. Virol. 2002; 76:8875–8889. [PubMed: 12163607]

Sok D, Doores KJ, Briney B, Le KM, Saye-Francisco KL, Ramos A, Kulp DW, Julien JP, Menis S, Wickramasinghe L, et al. Promiscuous Glycan Site Recognition by Antibodies to the High-Mannose Patch of gp120 Broadens Neutralization of HIV. Sci. Transl. Med. 2014; 6:236ra263.

Sok D, Laserson U, Laserson J, Liu Y, Vigneault F, Julien JP, Briney B, Ramos A, Saye KF, Le K, et al. The effects of somatic hypermutation on neutralization and binding in the PGT121 family of broadly neutralizing HIV antibodies. PLoS Pathog. 2013; 9:e1003754. [PubMed: 24278016]



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Sorzano CO, Bilbao-Castro JR, Shkolnisky Y, Alcorlo M, Melero R, Caffarena-Fernandez G, Li M, Xu G, Marabini R, Carazo JM. A clustering approach to multireference alignment of single-particle projections in electron microscopy. J. Struct. Biol. 2010; 171:197–206. [PubMed: 20362059]

Suloway C, Pulokas J, Fellmann D, Cheng A, Guerra F, Quispe J, Stagg S, Potter CS, Carragher B. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 2005; 151:41–60. [PubMed: 15890530]

van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 1996; 116:17–24. [PubMed: 8742718]

Voss NR, Yoshioka CK, Radermacher M, Potter CS, Carragher B. DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. J. Struct. Biol. 2009; 166:205–213. [PubMed: 19374019]

Walker LM, Huber M, Doores KJ, Falkowska E, Pejchal R, Julien JP, Wang SK, Ramos A, Chan-Hui PY, Moyle M, et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 2011; 477:466–470. [PubMed: 21849977]

Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009; 326:285–289. [PubMed: 19729618]



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Highlights

• Potent broadly neutralizing HIV antibody PGT124 contacts both glycan and

protein

• PGT124 contrasts with other family members by contacting only a single glycan

• Inferred germline antibody incorporates features important for protein and

glycan recognition

• Antibody maturation diversifies modes of glycan recognition



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Figure 1. Crystal structure of PGT124 in complex with HIV-1 gp120 and CD4(A) The ternary complex of PGT124 (purple and brown for heavy and light chains

respectively) and 2-domain CD4 (gray) bound to gp120 (light blue) is shown as a ribbon

representation. PGT124 contacts the N332 glycan and the base of V3. The glycan is

represented in ball-and-stick, with green carbons, red oxygens, and blue nitrogens, and

covered with its molecular surface. (B) CDR H3, L1, L2, L3 loops, and the light chain FR3

make extensive contacts with both the Man8 N332 glycan and V3 (in red) of gp120. (C) The

surface areas (Å2) buried on the gp120 protein and N332 glycan by individual CDR loops

and framework regions on PGT124 are presented on the lower right. (D) Fitting of the

PGT122-BG505 SOSIP.664 gp140 crystal structure into the PGT124:BG505 SOSIP EM

reconstruction at 17.8 Å resolution. The PGT122 fitting is excellent with a correlation of

0.9, indicating that PGT122 and PGT124 share a similar angle of approach for interaction

with Env (see also Figures S1 and S2 and Tables S1 and S6).



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Figure 2. Interaction between PGT124 and the base of the V3 loop(A) The interaction between PGT124 CDR loops (purple ribbons) and the base of the V3

loop (red ribbon). Red dots represent residues at the tip of the mini V3 loop that are not

visible in the electron density. The interaction with contacting residues is displayed in ball-

and-stick representation below. Contacts between side chains of the 324GDIR327 motif on

the V3 loop and the CDR loops of PGT124 are indicated with solid lines for van der Waals’

interactions and dotted lines for hydrogen bonds. (B) For comparison, the interaction

between PGT128 CDR loops (pale green) and the base of the V3 (gray) (PDB 3TYG) for

comparison with (A). The PGT128 antibody-antigen interactions with the V3 base are all H-

bonds of the Fab main chain with V3 main chain. (C) The V3 loop structures bound to

PGT124 or PGT128 are superimposed for comparison. (D) The effect on the neutralization

activity of PGT124 and PGT128 by single and double mutations of the V3 loop GDIR

motif. Values are shown as IC50 in μg/mL (see also Figures S3, S4 and S5).



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Figure 3. Interaction between PGT124 and the N332 glycan(A) Interaction of the CDR loops of PGT124 in purple with the oligomannose glycan at the

N332 position in red ball-and-stick representation with the D1, D2 and D3 arms labeled. (B) For comparison, the interaction of the PGT128 CDR loops (light green) is shown with the

N332 glycan (gray ball-and-stick representation). (C) The N332 glycan structures bound to

PGT128 or PGT124 are shown after superposition of the gp120 protein components.

Conformational differences are highlighted by comparative distances between equivalent

mannose moieties (D) Schematic diagram of the Man8GlcNAc2 glycan with the glycan

moiety nomenclature. (E) The effect on PGT124 neutralization activity of differences in

gp120 glycosylation arising from different cell expression systems and removal of the N332

glycan. Values are shown as IC50 in μg/mL (see also Figures S3, S5 and S6 and Tables S2

and S3).



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Figure 4. Epitope core of the PGT121 germline lineage(A) Cartoon representation of the interaction between mV3-gp120 core and the PGT124

variable region (left panel) and close-up views of the PGT124 epitope core with

the 324GDIR327 motif at the V3 base in blue spheres and the N332 glycan as pale green ball-

and-stick (middle and right panels). The PGT124 residues that contact the N332 glycan and

the V3 protein motif are represented in red and orange as a ball-and-stick, respectively, and

covered with its molecular surface. (B) Sequence alignment of heavy and light chains of

PGT121-124 Abs and germline precursors (* the germline CDR L3 and CDR H3 sequences

are not used for alignment because of ambiguity associated with D gene assignment). Red

and orange boxes indicate residues that contact the N332 glycan and the GDIR motif,

respectively, on the epitope core of PGT124, are also largely conserved in PGT121-123 and,

importantly, in the germline sequence (see also Figure S1).



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Figure 5. PGT122 contacts glycans at N137 and N301 but PGT124 accommodates these glycans without forming contacts(A) The PGT122 epitope is composed of V1 and V3 regions from gp120, as wells as four

glycans at N137, N156, N301 and N332. The CDR loops and FR3 are shown in cartoon

representation (in gray) (PDB ID:4NCO). (B) Superposition of PGT124 (in purple) on the

PGT122 epitope. (C) Although PGT124 seems capable of accommodating a glycan in the

open face formed by H2 and H3, those loops appear to be shifted towards the center of the

epitope, when compared to the PGT122 paratope, thereby avoiding contacts with the N137

glycan. (D) However, PGT124 is predicted to clash with the 301 glycan and presumably

pushes this glycan away to access the protein surface below.



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Figure 6. Evolution of recognition of complex glycans by the PGT121 family(A) Key residues on PGT121 (Kabat numbering) that contact the complex glycan attached at

N137 of gp120 V1 are represented in red as a stick-and-ball and covered with its molecular

surface on the ribbon representation of the antibody variable regions (left). Residues at

equivalent positions on PGT124 are highlighted in orange (right). (B) Sequence alignment

for heavy chain variable region for the germline precursor (GL) (* the germline CDR H3

sequence is not used for the alignment because of ambiguity associated with D gene

assignment), intermediate precursors (3H, 32H and 9H) and mature antibodies of the

PGT121 family (PGT121-124). Red boxes indicate residues involved in N137 glycan

binding by PGT121 while orange boxes indicate the residues at equivalent positions on

PGT124. (C) Glycan arrays illustrate that PGT124, in contrast to PGT121, PGT128 or

2G12, does not bind either complex or high mannose glycans with any demonstrable affinity

(see also Figures S1, S3 and S7).



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Figure 7. Summary of the contributions of the N137, N156, N301 and N332 glycans to recognition of the BG505 isolate by antibodies of the PGT121 lineageEarly precursors are highly dependent on the N301 and N332 glycans for neutralization, and

negatively impacted by the presence of the N137 glycan (* N137 glycan can play a double

role in glycan recognition: first it can inhibit antibody binding by shielding the epitope, here

represented in blue; second, although it can still inhibit antibody binding to some extent, the

N137 glycan can also be used by PGT121-3 to neutralize BG505 in the absence of N332,

here represented in blue/red). Affinity maturation appears to create two distinct lineages that

evolve separate dependencies on glycans that surround N332. N332 is the only critical

glycan in the PGT124 epitope, while the N137 glycan contributes negatively to recognition

by this antibody. Fold changes in neutralization IC50 for single and double glycan mutants

(Table S4) were used to determine whether a glycan positively contributes to the epitope

(decrease in neutralization IC50 upon removal) or contributes to shielding of the epitope

(increase in neutralization IC50 upon removal). A relative scale for both positive and

negative glycan contributions to the epitope was determined based on the extent of the

change in neutralization IC50. Interestingly, for PGT121-123, the N137 glycan contributes to

shielding in the presence of N332, but is a critical part of the epitope in the absence of N332

(see also Table S4 and S5).



NIH

-PA

Author M

anuscriptN

IH-P

A A

uthor Manuscript

NIH

-PA

Author M

anuscript

Role of Receptor Binding Specifity in Influenza A Virus Transmission and

Pathogenesis

Review

Role of receptor binding specificity in influenza Avirus transmission and pathogenesisMiranda de Graaf & Ron A M Fouchier*

Abstract

The recent emergence of a novel avian A/H7N9 influenza virus inpoultry and humans in China, as well as laboratory studies onadaptation and transmission of avian A/H5N1 influenza viruses,has shed new light on influenza virus adaptation to mammals.One of the biological traits required for animal influenza virusesto cross the species barrier that received considerable attention inanimal model studies, in vitro assays, and structural analyses isreceptor binding specificity. Sialylated glycans present on theapical surface of host cells can function as receptors for the influ-enza virus hemagglutinin (HA) protein. Avian and human influenzaviruses typically have a different sialic acid (SA)-binding prefer-ence and only few amino acid changes in the HA protein can causea switch from avian to human receptor specificity. Recent experi-ments using glycan arrays, virus histochemistry, animal models,and structural analyses of HA have added a wealth of knowledgeon receptor binding specificity. Here, we review recent data onthe interaction between influenza virus HA and SA receptors ofthe host, and the impact on virus host range, pathogenesis, andtransmission. Remaining challenges and future research prioritiesare also discussed.

Keywords A/H5N1 influenza virus; A/H7N9 influenza virus; airborne

transmission; pathogenesis; receptor binding specificity

DOI 10.1002/embj.201387442 | Received 19 November 2013 | Revised 17

February 2014 | Accepted 17 February 2014 | Published online 25 March 2014

EMBO J (2014) 33, 823–841

Influenza virus zoonoses and pandemics

Influenza A viruses can infect a wide range of hosts, including

humans, birds, pigs, horses, and marine mammals (Webster et al,

1992). Influenza A viruses are classified based on the antigenic

properties of the major surface glycoproteins hemagglutinin (HA)

and neuraminidase (NA). To date, 18 HA and 11 NA subtypes have

been described. All subtypes have been found in wild aquatic birds

except for the recently discovered H17N10 and H18N11 viruses,

which have only been detected in bats (Webster et al, 1992;

Fouchier et al, 2005; Tong et al, 2012, 2013). Throughout recent

history, avian-origin influenza viruses have crossed the species

barrier and infected humans. Some of these zoonotic events resulted

in the emergence of influenza viruses that acquired the ability to

transmit between humans and initiate a pandemic. Four pandemics

were recorded in the last century: the 1918 H1N1 Spanish pandemic,

the 1957 H2N2 Asian pandemic, the 1968 H3N2 Hong Kong

pandemic, and the 2009 H1N1 pandemic (pH1N1) that was first

detected in Mexico (reviewed in Sorrell et al, 2011). Various other

influenza A viruses of pig and avian origin (e.g., of subtypes H5,

H6, H7, H9, and H10) have occasionally infected humans—some-

times associated with severe disease and deaths—but these have

not become established in humans.

The H5N1 avian influenza virus that was first detected in Hong

Kong in 1997 has frequently been reported to infect humans and

cause serious disease. As of October 8, 2013, WHO has been

informed of 641 human cases of infection with H5N1 viruses, of

which 380 died (www.who.int/influenza/human_animal_interface/

en/). The majority of these cases occurred upon direct or indirect

contact with infected poultry. Due to the high incidence of zoonotic

events, the enzootic circulation of the virus in poultry, and the

severity of disease in humans, the H5N1 virus is considered to pose

a serious pandemic threat. Fortunately, sustained human-to-human

transmission has not been reported yet (Kandun et al, 2006; Wang

et al, 2008).

A more recent example of a major zoonotic influenza A virus

outbreak started in China in early 2013. This outbreak, caused by

an avian H7N9 virus, resulted in 137 laboratory-confirmed human

cases of infection and 45 deaths (www.who.int/influenza/human_

animal_interface/en/). Although one case of possible human-to-

human transmission was described, thus far, this outbreak also has

not lead to sustained transmission between humans (Gao et al,

2013b; Qi et al, 2013). Some mild cases of infection were reported,

but the Chinese H7N9 influenza viruses frequently caused severe

illness, characterized by severe pulmonary disease and acute respi-

ratory distress syndrome (Gao et al, 2013a,b). Although influenza

viruses of the H7 subtype have sporadically crossed the species

barrier in the past, with outbreaks reported, for example, in the

United Kingdom, Centers for Disease Control and Prevention

(2004), Canada (Tweed et al, 2004), the Netherlands (Fouchier

et al, 2004), and Italy (Puzelli et al, 2005; www.who.int/influenza/

human_animal_interface/en/), the 2013 H7N9 virus appeared to

Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands*Corresponding author. Tel: +31 10 7044067; Fax: +31 10 7044760; E-mail: [email protected]

ª 2014 The Authors The EMBO Journal Vol 33 | No 8 | 2014 823

www.who.int/influenza/human_animal_interface/en/






jump the species barrier more easily and was generally associated

with more serious disease in humans. Interestingly, the internal

genes of the H7N9 virus belong to the same genetic lineage as the

internal genes of the H5N1 virus; both are derived from H9N2

viruses (Guan et al, 1999; Lam et al, 2013). The H7N9 virus is

thought to have emerged upon four reassortment events between

H9N2 viruses and avian-origin viruses of subtype H7 and N9 (Lam

et al, 2013). The H9N2 virus itself has been shown to have zoonotic

potential as well, resulting in relatively mild human infections upon

contact with poultry. H9N2 viruses remain enzootic in domestic

birds in many countries of the Eastern Hemisphere (Peiris et al,

1999).

For all influenza pandemics of the last century, the animal-

origin viruses acquired the ability to transmit efficiently via

aerosols or respiratory droplets (hereafter referred to as “airborne

transmission”) between humans (Sorrell et al, 2011). To evaluate

airborne transmission in laboratory settings, ferret and guinea pig

transmission models were developed, in which cages with unin-

fected recipient animals are placed adjacent to cages with infected

donor animals. The experimental setup was designed to prevent

direct contact or fomite transmission, but to allow airflow from

the donor to the recipient ferret (Lowen et al, 2006; Maines et al,

2006). Using the ferret transmission model, pandemic and

epidemic viruses isolated from humans are generally transmitted

efficiently via the airborne route, whereas avian viruses are

generally not airborne transmissible (Sorrell et al, 2011; Belser

et al, 2013b).

Why some animal influenza viruses frequently infect humans,

whereas others do not, and how viruses adapt to become airborne

transmissible between mammals have been key questions in influ-

enza virus research over the last decade. Studies on H1N1, H2N2,

and H3N2 viruses from the 1918, 1957, and 1968 pandemics,

respectively, revealed that interaction of HA with virus receptors

on host cells was a critical determinant of host adaptation and

airborne transmission between ferrets (Tumpey et al, 2007; Pappas

et al, 2010; Roberts et al, 2011). Sorrell and colleagues showed that

a wild-type avian H9N2 virus was not airborne transmissible in

ferrets, but reassortment with a human H3N2 virus and subsequent

adaptation in ferrets yielded an airborne transmissible H9N2 virus,

primarily due to changes in the H9N2 HA and NA surface glyco-

proteins (Sorrell et al, 2009). Several laboratories have studied the

transmissibility of avian H5N1 viruses and their potential to

become airborne, and four studies recently described airborne

transmission of laboratory-generated H5N1 influenza viruses.

Three of these studies—two using ferrets and one using guinea

pigs—used H5 reassortant viruses between human pH1N1 or H3N2

viruses and H5N1 virus (Chen et al, 2012; Imai et al, 2012; Zhang

et al, 2013d). Herfst et al (2012)were the first to show mammalian

adaptation of a fully avian H5N1 virus to yield an airborne trans-

missible virus in ferrets. Although avian H7 influenza viruses are

generally not transmitted via the airborne route between ferrets

(Belser et al, 2008), H7N9 strains from the Chinese 2013 outbreak

were shown to be transmitted via the airborne route without

further adaptation, albeit less efficiently as compared to human

seasonal and pandemic viruses (Belser et al, 2013a; Richard et al,

2013; Watanabe et al, 2013; Xu et al, 2013; Zhang et al, 2013a;

Zhu et al, 2013). No transmission was observed from pigs to

ferrets (Zhu et al, 2013).

It has thus become increasingly clear that animal influenza

viruses beyond the H1, H2, and H3 subtypes—specifically H5, H7,

and H9—can acquire the ability of airborne transmission between

mammals. To date, the exact genetic requirements for animal

influenza virus to cross the species barrier and establish efficient

human-to-human transmission remain largely unknown, but

receptor binding specificity is clearly one of the key factors.

HA as determinant of receptor binding specificity

As a first step to entry and infection, influenza viruses attach with

the HA protein to sialylated glycan receptors on host cells. The influ-

enza virus HA protein is a type I integral membrane glycoprotein,

with a N-terminal signal sequence. Post-translational modifications

include glycosylation of the HA and acylation of the cytoplasmic tail

region. Cleavage of the HA (HA0) by cellular proteases generates

the HA1 and HA2 subunits, which form a disulfide bond-linked

complex. The HA protein forms trimers, with each monomer

containing a receptor binding site (RBS) capable of engaging a sialy-

lated glycan receptor, a vestigial esterase subdomain, and a fusion

subdomain (Fig 1).

Hirst (1941) was the first to demonstrate the ability of influenza

viruses to agglutinate and elute from red blood cells. Treatment of

these cells with Vibrio cholerae neuraminidase (VCNA) revealed that

the ability to agglutinate and elute was dependent on sialic acid (SA;

Gottschalk, 1957). Early comparisons of influenza viruses isolated

from different species revealed differences in their abilities to agglu-

tinate red blood cells from various species. Using red blood cells

and cells expressing only a certain type of SA, it was discovered that

human and animal influenza viruses displayed differences in the

receptor specificity of HA (Rogers & Paulson, 1983). Avian influenza

viruses were found to have a preference for SA that are linked to the

galactose in an a2,3 linkage (a2,3-SA; Rogers & Paulson, 1983;

Nobusawa et al, 1991). In contrast, human influenza viruses (H1,

H2, and H3) attached to SA that are linked to galactose in an a2,6linkage (a2,6-SA; Gambaryan et al, 1997; Matrosovich et al, 2000).

Pig influenza viruses either attached to both a2,3-SA and a2,6-SA,or exclusively to a2,6-SA (Rogers & Paulson, 1983). These gross

differences in receptor binding properties were found to be impor-

tant determinants of virus host range and cell and tissue tropism. A

wide range of assays has been developed to assess HA binding

specificity and affinity to glycans containing a2,3-SA and a2,6-SA(Paulson et al, 1979; Sauter et al, 1989; Takemoto et al, 1996;

Stevens et al, 2006a; Chutinimitkul et al, 2010b; Lin et al, 2012;

Matrosovich & Gambaryan, 2012). Binding assays such as glycan

arrays, which allow the investigation of hundreds of different sialy-

lated glycan structures, have demonstrated that influenza virus

receptor specificity also involves structural modifications of the SA

and overall glycan (Gambaryan et al, 2005; Stevens et al, 2006b).

Influenza virus receptors

Types of glycan structuresMammalian cells are covered by a glycocalyx, which consists of

glycolipids, glycoproteins, glycophospholipid anchors and proteogly-

cans (Varki & Varki, 2007; Varki & Sharon, 2009). Glycans represent

The EMBO Journal Vol 33 | No 8 | 2014 ª 2014 The Authors

The EMBO Journal Role of HA in influenza A virus transmission Miranda de Graaf & Ron A M Fouchier

824

one of the fundamental building blocks of life and have essential

roles in numerous physiological and pathological processes

(reviewed in Hart, 2013). Glycans that are exposed on the exterior

surface of cells play an important role in the attachment of toxins

and pathogens like influenza A viruses.

Several types of glycans exist, including N-glycans, O-glycans,

and glycolipids (reviewed in Brockhausen et al, 2009; Schnaar et al,

2009; Stanley et al, 2009). The N-glycans can be further divided into

different types such as “complex,” “hybrid,” and “oligomannose.”

There is a wide variety of O-glycans, with eight different core struc-

tures. Cores 1 and 2 are common structures that are present on

glycoproteins (Fig 2). This particular classification of glycan struc-

tures is not necessarily relevant for binding of influenza viruses. For

O-glycans and N-glycans, there is extensive structural diversification

at the termini of glycan chains, which is potentially more important

than differences in the core structures.

Receptor-mediated entry of influenza viruses is reduced in the

absence of sialylated N-glycans (Chu & Whittaker, 2004; de Vries

et al, 2012). However, specific roles of sialylated N-glycans,

O-glycans, and glycolipids for binding and entry of influenza viruses

are not fully understood. Mouse cells that are deficient in the

production of glycolipids can be infected efficiently with human

H3N2 virus, indicating that sialylated glycolipids are not essential

for infection (Ichikawa et al, 1994; Ablan et al, 2001). Hamster cells

that are deficient in the production of sialylated N-glycans cannot be

efficiently infected with influenza viruses in the presence of serum

(Robertson et al, 1978; Chu & Whittaker, 2004). However, when the

production of sialylated N-glycans was restored, H1N1 virus was

able to bind and infect these cells (Chu & Whittaker, 2004).

Furthermore, it was shown that internalization of influenza via

macropinocytic endocytosis is dependent on N-glycans, but that for

clathrin-mediated endocytosis, N-glycans are not required (de Vries

et al, 2012). It should be noted that most studies are based on in

vitro work with human viruses; whether the same principles hold

true in vivo and for avian viruses is not known.

Types of sialic acidSAs share a nine-carbon backbone and are among the most diverse

sugars found on glycan chains of mammalian cell surfaces.

Common SAs found in mammals are N-acetylneuraminic acid

(Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc; Fig 3; reviewed

in Varki & Varki, 2007). Neu5Gc has been detected in pigs,

monkeys, and a number of bird species, but humans and some

avian species like chickens or other poultry, do not contain Neu5Gc

or only minor quantities (Chou et al, 1998; Muchmore et al, 1998;

Schauer et al, 2009; Walther et al, 2013).

Lectins Maackia amurensis agglutinin (MAA) and Sambucus

nigra agglutinin (SNA) recognize a2,3-SA and a2,6-SA, respectively,and are often used to study the distribution and expression of influ-

enza virus receptors in tissues and cells. There are two different

isotypes of MAA: MAA-1 (also known as MAL or MAM) and MAA-2

(also known as MAH). MAA-1 binds primarily to SAa2,3Galb1-4Glc-NAc that is found in N-glycans and O-glycans. MAA-2 preferentially

binds to SAa2,3Galb1-3GalNAc, which is found in O-glycans. Unfor-

tunately, both MAA-1 and MAA-2 also bind strongly to sulfated

glycan structures without SA. The exact specificities of MAA-1 and

MAA-2 are reviewed comprehensively by Geisler and Jarvis (2011).

To determine which glycan structures present a2,3-SA and a2,6-SA,MAA and SNA can be used in combination with the lectins Conca-

navalin and Jacalin to detect N-glycan and O-glycan structures,

respectively. Unfortunately, lectin staining provides little informa-

tion on the differences in overall glycan structures, including the

number of “antennas”, fucosylation, and the presence of N-acetyllac-

tosamine repeats, all of which can influence influenza virus receptor

binding. This encouraged scientists to elucidate the glycome of the

respiratory tissue of several influenza virus host species.

SA receptor distribution in humans, pigs, birds, and ferretsIn humans, a2,6-SAs are expressed more abundantly in the upper

respiratory tract compared to the lower respiratory tract (Fig 4). In

the nasopharynx of humans, a2,6-SA and a2,3-SA are detected on

ciliated cells as well as mucus-producing cells. In the bronchus,

Thr-160-Ala

LSTc

Fusion peptide

Vestigialesterasesubdomain

Gly-228-Ser

His-110-Tyr

Thr-318-Ile

Asn-158-Asp

Gln-226-Leun-226-Leu

Asn-224-Lys

RBS

Figure 1. Cartoon representation of the hemagglutinin structure of anH5N1 influenza A virus (protein database code 4KDO, representing amutant version of A/Vietnam/1203/04).The human receptor analog, LSTc (pink) is docked into the receptor binding site;the vestigial esterase subdomain and fusion peptide are depicted in yellow andorange, respectively. The amino acid substitutions described by Herfst et alare shown in red, and the mutations of Imai et al are shown in blue.Substitution Gln-226-Leu was described by both groups (Herfst et al, 2012;Imai et al, 2012).

ª 2014 The Authors The EMBO Journal Vol 33 | No 8 | 2014

Miranda de Graaf & Ron A M Fouchier Role of HA in influenza A virus transmission The EMBO Journal

825

a2,3-SA and a2,6-SA are heterogeneously distributed in the

epithelium with no clear distinction between ciliated and non-

ciliated cells. Bronchial epithelium contained a higher percentage of

a2,3-SA compared to a2,6-SA (Nicholls et al, 2007a). Staining for

a2,3-SA (MAA-II) was most abundant in cells lining the alveoli,

most likely type II cells (Shinya et al, 2006; Nicholls et al, 2007a).

The respiratory tract of young children expresses more a2,3-SA and

a lower level of a2,6-SA compared to adults (Nicholls et al, 2007a).

To identify the structures of sialylated glycans present in the

human respiratory tract, glycomic analysis of human bronchus,

lung, and nasopharynx was performed (Walther et al, 2013).

Heterogeneous mixtures of bi-, tri-, and tetra-antennary N-glycans

with varying numbers of N-acetyllactosamine repeats were

detected, as well as significant levels of core fucosylated struc-

tures and high mannose structures. Compared to the glycan

composition of the bronchus and lung, the nasopharynx displayed

a smaller spectrum of glycans, with fewer N-acetyllactosamine

repeats. Both a2,6-SA and a2,3-SA receptors were detected, but

with a higher percentage of a2,3-SA receptors in the bronchus

compared to the lungs. Sialylated core-1 and core-2 O-glycans

were present.

The glycan composition of the human respiratory tract was also

compared with the glycans present on available glycan arrays

(Table 1). The O-glycan composition of the human respiratory tract

was represented well on glycan arrays, but the complex N-glycan

structures were underrepresented. On the other hand, a large

number of glycans that are present on the array were not detected

in the human respiratory tract and can therefore be largely ignored

when studying human influenza virus receptor specificity.

Pigs are often regarded as a potential “mixing vessel” of human

and avian influenza viruses, because pigs express both a2,3-SA and

a2,6-SA in the respiratory tract (Scholtissek, 1990; Ito et al, 1998;

Nelli et al, 2010; Van Poucke et al, 2010; Fig 4). Given the recent

data on SA presence in the human respiratory tract (see above), this

role as a mixing vessel may not be unique to pigs. The a2,6-SAreceptors were dominant in the ciliated epithelia along the upper

respiratory lining of the trachea and bronchus. There was a gradual

increase in a2,3-SA receptors toward the lower respiratory lining.

The mucosa of the respiratory tract predominantly contained

a2,3-SA, with a2,6-SA mainly confined to mucous/serous glands.

The a2,3-SA receptors were more highly expressed in the epithelial

lining of the lower respiratory tract (bronchioles and alveoli; Nelli

et al, 2010).

Primary pig respiratory epithelial cells, trachea, and lung tissue

were described to contain high mannose structures and complex

glycans with core fucosylated bi-, tri-, and tetra-antennary struc-

tures. In the pig respiratory epithelial cells, structures bearing

N-acetyllactosamine repeats were expressed, but there was a

relative lack of these structures in pig trachea and lung tissue

(Table 1). Both Neu5Gc and Neu5Ac were present, with a higher

abundance of the latter (Bateman et al, 2010; Sriwilaijaroen et al,

2011; Chan et al, 2013b). Pig lung contains both core 1 and core 2

O-glycan structures. For N-glycans, there was a higher abundance

Ser/Thr Ser/Thr

Core 1 Core 2

O-glycans

Cer

GlycolipidN-glycans

Complex

Oligomannose

Asn

LN

LN

Asn Asn

Hybrid

Figure 2. N-glycans, O-glycans, and glycolipids.Examples of three types of N-glycans are shown; complex with multiple N-acetyllactosamine repeats (LN), oligomannose, and hybrid (with the common core boxed). Aglycolipid and two types of O-glycans are also shown; core 1 (in box) and core 2 (in box) O-glycans. Monosaccharides are depicted using the following symbolicrepresentations: fucose (red triangle), galactose (yellow circle), N-acetyl glucosamine (blue square), N-acetyl galactosamine (yellow square), glucosamine (blue circle),mannose (green circle), sialic acid (purple diamond).

OHOH

OH

HO

HO

HN

R

O

O

COOHR = –CH

3

R = –CH2OH

Neu5Ac

Neu5Gc

Figure 3. Structures of Neu5Aca and Neu5Gc.



826

of a2,6-SA in the lungs and trachea compared to a2,3-SA (Chan

et al, 2013b). The pig respiratory tract presents a smaller diversity

of sialylated N-glycan structures compared to the glycome of the

human respiratory tract (Sriwilaijaroen et al, 2011; Walther et al,

2013).

Avian species have a2,3-SA and a2,6-SA in both the respiratory

and intestinal tract, although there are differences in the abundance

of these receptors between different species (Franca et al, 2013).

Chickens and common quails express a2,3-SA and a2,6-SA on a

large range of different epithelial cells in the respiratory tract and

intestine (Nelli et al, 2010; Trebbien et al, 2011; Costa et al, 2012;

Fig 4). There is conflicting data for the presence of a2,6-SA in the

intestine of mallards: Some groups found expression of a2,6-SA,whereas others did not (Kuchipudi et al, 2009; Costa et al, 2012;

Franca et al, 2013).

Chicken red blood cells are frequently used for influenza virus

agglutination assays. Analysis of its glycome revealed that chicken

red blood cells present a plethora of N-glycans, both di-, tri-, and

tetra-antennary glycan structures with a mixture of a2,3-SA and

a2,6-SAs, but compared to the glycome of the human respiratory

tract, an absence of repeating N-acetyllactosamine units was

observed (Table 1).

Ferrets are widely used as an animal model for influenza virus

infections because they have a similar distribution of a2,6-SA and

a2,3-SAs throughout the respiratory tract as humans (Leigh et al,

1995; Kirkeby et al, 2009). In the ferret trachea and bronchus,

a2,6-SA was abundantly expressed by ciliated cells and submucosal

glands (Fig 4). The presence of a2,3-SA was detected in the lamina

propria and submucosal areas, but not on ciliated cells. Alveoli

expressed both a2,3-SA and a2,6-SA although staining for the latter

was more abundant (Jayaraman et al, 2012).

Structural determinants of receptor specificity

In 1981, the first crystal structure of influenza A virus HA was

described by Wilson et al (1981). These and other structural studies

showed that HA is expressed as a trimer, with a site for SA binding

in the membrane-distal tip of each of the monomers. Each site

comprises a pocket of conserved amino acids that is edged by the

190-helix, the 130-loop, and the 220-loop (Fig 5A). A set of

conserved residues, 98-Tyr, 153-Trp, 183-His, and 195-Tyr (num-

bering throughout the manuscript is based on H3 HA), forms the

base of the RBS (Skehel & Wiley, 2000; Ha et al, 2001). Although

sequence differences in the 190-helix, the 130-loop, and the

220-loop exist between viruses of different subtypes and from

different hosts (Fig 5B), the conformation of each of these elements

is similar. Structural studies have demonstrated that the RBS of

human and pig influenza viruses that bind a2,6-SA is “wider” than

the RBS of avian influenza viruses (Ha et al, 2001; Stevens et al,

2004).

To study interactions of HA with receptors, HAs are crystallized

and soaked in human or avian receptor analogs. Commonly, linear

sialylated pentasaccharides LSTc (a2,6-SA) and LSTa (a2,3-SA) are

used as human and avian receptor analogs, respectively (Fig 6).

Among the major difficulties of structural studies are the flexible

++++++

α2,6 SA

++++++

α2,3 SAMAA-I

++++++

α2,3 SAMAA-II

Figure 4. Overall distribution of a2,6-sialic acid (SA; red stars) and a2,3-SA based on Maackia amurensis agglutinin (MAA)-I (blue stars) and MAA-II staining (yellowstars) in the epithelium of the respiratory tract (nasal cavity, trachea, bronchus, bronchiole, and alveoli) of humans (Nicholls et al, 2007a), ferrets (Xu et al, 2010a), pigs(Nelli et al, 2010), and chicken, and the small and large intestine of chickens (Costa et al, 2012). The size of the star shape corresponds with the relative receptorabundance. Receptor distribution may vary between studies, possibly due to the lectins used, experimental methods, or real variation in receptor distribution betweenanimals. Lectin staining was not performed for the human trachea, nasal cavity of pigs and nasal cavity and bronchioles of ferrets in the cited studies. No MAA-I stainingwas performed for the chicken tissues. Figure based on Herfst et al, 2012.



827

Table

1.Glyca

nstructuresof

thehuman

resp

iratorytrac

tthat

arerepresentedon

glycan

arrays

(adap

tedfrom

Walther

etal,2

013

)

Glyca

narrays

Human

Ck

Pig

Array

AArray

BArray

CArray

DArray

E

a2,6

a2,3

Both

a2,6

a2,3

Both

a2,6

a2,3

Both

a2,6

a2,3

Both

a2,6

a2,3

Both

Lung

Bronch

us

Tonsil

andNP

RBC

Lung

√√

√√

√√

√

√√

√√

√√

√√

√√

√√

√

√√

√√

√√

√√

√√

√√

√√

√√

√√

√√

√√

√√

√√

√

√√

√√

√√

√√

√

√√

√√

√√

√nd

√√

√√

√√

√√

nd

√

√√

√√

√√

√nd

√√

√√

√nd

√√

√nd

Glycanstructuresisolated

from

tissues

ofthehuman

respiratorytract(nasop

haryn

x,bron

chus,an

dlungs)werecompa

redwithglycan

sprintedon

theglycan

arrays

oftheconsortium

offunctional

glycom

ics

(version

5)(A),Cen

terforDisease

Con

trol

andPreven

tion

(B),(C)Child

set

al(2009),W

onget

al(D;Z

han

get

al,2008)

and(E)de

Vrieset

al(2014

).Th

e√symbo

lineach

columnindicatesthat

theglycan

ispresen

ton

therespective

arrayor

tissue.Glycanstructuresdetected

inthepiglung(Chan

etal,2013

b),andchicken(Ck)

redbloo

dcells

(Aichet

al,2011

),that

werebo

thpresen

tin

thehuman

respiratorytract

andrepresen

tedby

oneor

multiple

arrays

wereincluded.



828

nature of both the HA protein and the glycan structure, which is

largely undetectable in crystallography, and the lack of knowledge

of the SA receptors that are relevant for infection in vivo (see

above). LSTa in complex with the HA of avian H1, H2, H3, and H5

viruses is usually bound in a trans conformation of the a2,3 linkage.

In contrast, LSTc is bound in a cis conformation of the a2,6 linkage

by HA of H1, H2, and H3 human influenza viruses (Gamblin &

Skehel, 2010; Fig 6A).

HA of pandemic influenza virusesThe H2N2 and H3N2 pandemics were caused by viruses containing

HAs of avian origin (Scholtissek et al, 1978; Kawaoka et al, 1989).

Two mutations, Gln-226-Leu and Gly-228-Ser in the RBS, were suffi-

cient for avian H2 and H3 viruses to switch from a2,3-SA to a2,6-SAspecificity (Connor et al, 1994; Matrosovich et al, 2000; Fig 5B).

The Leu at position 226 caused a shift of the 220-loop, resulting in a

widening of the RBS, favorable binding to the human receptor, and

abolished binding to the avian receptor (Liu et al, 2009; Xu et al,

2010b). This shift and the widening of the RBS were maximal if both

226-Leu and 228-Ser were present. In contrast to human H2 and H3,

the HAs of human H1N1 viruses retained 226-Gln and 228-Gly, but

bound a2,6-SA nevertheless (Rogers & D’Souza, 1989). The a2,6-SApreference of human H1N1 virus was found to be determined

primarily by 190-Asp and 225-Asp (Matrosovich et al, 2000; Glaser

et al, 2005). Structural differences in the 220-loop resulted in a

lower location of 226-Gln, enabling H1 HAs to bind the human

receptor analog despite the presence of 226-Gln (Gamblin et al,

2004). Upon binding to the human receptor, interactions between

the receptor and 222-Lys and 225-Asp in the 220-loop are formed,

and a hydrogen bond with 190-Asp (Gamblin et al, 2004; Zhang

et al, 2013c).

HA of airborne H5N1 influenza virusesImai et al (2012) reported airborne transmission of a pH1N1 reas-

sortant virus that contained the H5 HA of A/Vietnam/1203/04

(VN1203) with amino acid substitutions Asn-224-Lys and Gln-226-

Leu, known to result in a switch in receptor specificity, Asn-158-

Asp that resulted in the removal of a glycosylation site, and Thr-

318-Ile in the stalk region which stabilized the trimer structure

(Fig 1). The effect of these substitutions on receptor binding and

HA structure was investigated in the context of autologous

VN1203 and the closely related A/Vietnam/1194/04 (VN1194) HA

(Lu et al, 2013; Xiong et al, 2013a). Xiong et al reported a small

increase in binding to a2,6-SA for VN1194 with the mutations

described by Imai et al (VN1194mut), similar to what was

described in the original work (Imai et al, 2012). Both VN1194mut

and VN1203mut displayed a substantially decreased affinity for

a2,3-SA. Overall, the affinity for human and avian receptors of the

mutant H5 HA was 5- to 10-fold lower than for human H3 HA

(Xiong et al, 2013a). VN1194mut HA acquired the ability to bind

a2,6-SA in the same cis conformation like human H1, H2, and H3

HAs. In particular, the Gln-226-Leu substitution facilitated binding

to a2,6-SA, while restricting binding to a2,3-SA. For both the

VN1203mut and VN1194mut HA, the RBS between the 130- and

220-loops had increased in size by approximately 1–1.5 A. It was

further suggested that the glycosylation at residue 158 might steri-

cally block HA binding to cell surface SAs (Lu et al, 2013; Xiong

et al, 2013a).

A B

190 225226

228

130 140 220 230190 199

130-loop 190-helix 220-loop

H1A/commonteal/NL/10/2000 (Av) ETTKGVTAACS EQQTLYQNT RPKVRGQAGRM

A/Memphis/7/2001 (Hu) HTVTGVSASCS DQRALYHTE RPKVRDQEGRI

A/SC/1/1918 (Hu) ETTKGVTAACS DQQSLYQNA RPKVRDQAGRM

A/NL/602/2009 (Hu) DSNKGVTAACP DQQSLYQNA RPKVRDQEGRM

A/Sw/Ind/P12439/2000 (Pi) DTNRGVTAACP DQQSLYQNA RPKVRDQAGRI

H3A/Mallard/SW/50/2000 (Av) VTQNGGSNACK EQTNLYVQA RPWVRGQSGRI

A/HK/01/1968 (Hu) VTQNGGSNACK EQTSLYVQA RPWVRGLSSRI

H2A/Mallard/NL/5/1999 (Av) TTT–GGSRACA EQRTLYQNV RPKVNGQGGRM

A/NL/056H1/1960 (Hu) TTT–GGSRACA EQRTLYQNV RPEVNGLGSRM

H5A/Indo/05/2005 (Av) EASSGVSSACP EQTRLYQNP RSKVNGQSGRM

A/Indo/05/2005 (Tr) EASSGVSSACP EQTRLYQNP RSKVNGLSSRM

H7A/Mallard/NL/12/2000 (Hu) IRTNGATSACR EQTKLYGSG RPQVNGQSGRI

A/NL/219/2003 (Av) IRTNGTTSACR EQTKLYGSG RPQVNGQSGRI

A/Shanghai/02/2013 (Av) IRTNGATSACR EQTKLYGSG RPQVNGLSGRI

220-loop

130-loop

190-helix

195-Tyr

153-Trp

183-His

98-Tyr

Figure 5. Cartoon representation of a human influenza hemagglutinin (database code 2YP4) receptor binding site with the 130-loop, 190-helix, and 220-loop and theconserved residues, 98-Tyr, 153-Trp, 183-His, and 195-Tyr in complex with the human receptor analog LSTc in cis conformation (A). Amino acid alignment of the 130-loop,190-helix, and 220-loop of human (Hu), avian (Av), pig (Pi) influenza viruses, and an airborne transmissible H5N1 virus (Tr) (B). The sequences are derived from thefollowing virus strains: A/Teal/NL/10/2000 (CY060178), A/Memphis/7/2001 (CY020149), A/SC/1/1918 (F116575), A/Mallard/SW/50/2000 (CY060308), A/HK/01/1968(CY112249), A/Mallard/NL/5/1999 (CY064950), A/NL/056H1/1960 (CY077786), A/Indo/5/2005 (CY116646), airborne transmissible A/Indo/05/2005 (CY116686), A/Mallard/12/2000 (GU053030), A/NL/219/2003 (AY338459) and A/Shanghai/02/2013 (KF021597).



829

Herfst et al (2012) described fully avian airborne transmissible

H5N1 viruses based on A/Indonesia/5/05 (INDO5), of which the

HA consistently contained substitution His-110-Tyr with a so far

unknown effect, Thr-160-Ala that removes the same glycosylation

site as Asn-158-Asp, and Gln-226-Leu and Gly-228-Ser that were

shown to affect receptor specificity (Fig 1). The effect of these

substitutions on receptor binding and HA structure was also investi-

gated (INDO5mut). INDO5mut had a approximately ninefold

reduced affinity for a2,3-SA and a > threefold increased affinity for

a2,6-SA compared to wild-type HA as measured by surface plasmon

resonance. Overall, the affinity for avian and human receptors of

the INDOmut HA was still relatively low. INDO5 bound LSTc in

trans conformation, which is different from human HAs. INDO5mut

bound this human receptor analog in cis conformation, in an orien-

tation similar as for pandemic viruses of other virus subtypes. The

226-Leu created a favorable environment for the non-polar portion

of the human receptor analog and makes a tighter interaction

between the RBS and receptor analog. Substitutions Gln-226-Leu

and Gly-228-Ser resulted in a RBS that was approximately 1 A wider

between the 130- and 220-loops compared to that of wild-type HA,

as reported for human HAs (Zhang et al, 2013b).

Collectively, these studies on receptor binding and structure of

HA of airborne transmissible H5 viruses thus showed a binding pref-

erence for human over avian receptors (although with overall low

affinity), widening of the RBS as a consequence of Gln-226-Leu, and

binding to the human receptor analog LSTc in a similar conforma-

tion as shown for human pandemic HAs.

Both airborne H5 HAs and a third one described in Zhang et al

lost the same glycosylation site near the RBS that was associated

with increased binding affinity, potentially as a result of reduced

steric hindrance of interactions between HA and the receptor (Herfst

et al, 2012; Imai et al, 2012; Zhang et al, 2013d). H5 and H7 influ-

enza viruses from domestic birds generally have more glycosylation

sites on the globular head than wild-bird influenza viruses, which

has been associated with reduced binding affinity (Matrosovich

et al, 1999). The glycosylation state of HA was previously shown to

affect both receptor binding efficiency and immunogenicity (Skehel

et al, 1984; Schulze, 1997). During the evolution of influenza

viruses, glycosylation sites are removed or introduced, resulting in

evasion of host immune responses. In addition, changes in glycosyl-

ation may cause functional differences, for example, by affecting the

stability of the RBS. Removal of N-glycans by means of glycosidase

F treatment was shown to enhance hemadsorbing activity of HA

(Ohuchi et al, 1997). The importance of proper glycosylation is

further evident from studies on recombinant soluble HA trimers.

Different expression systems can result in differences in the glyco-

sylation of the HA, and the presence of, for example, sialylated

N-glycans on HA can narrow its receptor binding range, which

can be increased upon the removal of SA by the addition of exoge-

nous NA (de Vries et al, 2010). Thus, differences in glycosylation of

the HA can have major implications for receptor specificity and

affinity.

HA of H7N9 influenza viruses

During the outbreak in China in 2013, several residues that are asso-

ciated with changes in the receptor specificity were observed in HA

of the circulating H7N9 viruses. A/Shanghai/1/13 contained 138-

Ser, which was shown previously to enhance binding to a2,6-SAof pig H5N1 viruses (Nidom et al, 2010). A/Anhui/1/13 and A/

Shanghai/2/13 possessed 226-Leu, which enhances binding to

a2,6-SA in H7 avian influenza viruses, and 186-Val, which was also

reported to influence receptor binding of H7 (Gambaryan et al,

2012; Srinivasan et al, 2013; Yang et al, 2013). However, none of

the H7N9 viruses contained 228-Ser. The H7 genetic lineage from

which the H7N9 HA was derived naturally lacks a glycosylation site

at the tip of HA, the one that was lost in the airborne H5 viruses.

Glc5

Glc5

Gal4

Gal4

Gal2

Gal2

Sia1

Sia1

GlcNAc3

GlcNAc3

LSTa LSTc

LSTa LSTc

Glc5Gal4Gal2Sia1

α2,3 β3 β3 β4

GlcNAc3 Glc5Gal4Gal2

α2,6 β4 β3 β4

Sia1 GlcNAc3

A

B

Figure 6. Human and avian receptor analogs in free conformation (Figure adapted from Xu et al, 2009).Avian receptor analog LSTa is shown in trans conformation and the human receptor analog LSTc in cis conformation (A). Symbolic representation of the LSTa and LSTcstructures (B), with the monosaccharides sialic acid (purple diamond), galactose (yellow circle), N-acetylglucosamine (blue square) and glucose (blue circle).



830

Multiple research groups have tested the receptor specificity of

H7N9 viruses, most of which reported dual receptor specificity

(Ramos et al, 2013; van Riel et al, 2013; Shi et al, 2013; Watanabe

et al, 2013; Xiong et al, 2013b; Zhou et al, 2013). Overall, A/Anhui/

1/2013 was found to bind better to a2,6-SA than A/Shanghai/1/

2013. In glycan arrays, A/Anhui/1/2013 bound to bi-antennary

structures as well as structures containing long N-acetyllactosamine

repeats (Belser et al, 2013a; Watanabe et al, 2013). H7N9 influenza

viruses in general had higher affinity for a2,6-SA compared to other

H7 influenza viruses. However, in most studies, the H7N9 viruses

maintained preference for avian over human receptors, which is in

contrast to the pandemic and airborne transmissible H5 viruses.

The H7N9 HA structure of A/Anhui/1/2013 in complex with

human and avian receptors was solved and compared with those of

avian H7 HAs. Both LSTc and LSTa were bound to HA in a cis

conformation (Shi et al, 2013; Xiong et al, 2013b). LSTc was shown

to exit the RBS of H7N9 HA in a different direction from that seen

with all pandemic virus HAs and the airborne transmissible H5 HA.

The H7 HAs have a subtype-specific insertion in the 150-loop,

causing this loop to protrude closer to the RBS. The function of this

150-loop is not yet known (Russell et al, 2006). The Gln-226-Leu

substitution in H7N9 HA provided a non-polar-binding site accom-

modating the human receptor, and substitution Gly-186-Val

increased the hydrophobicity of the binding site to further favor

interactions with this receptor. The space between the 220-loop and

the 130-loop was widened in H7N9 HA, but only marginally so,

compared to pandemic virus HA and airborne H5 HA (Xiong et al,

2013b).

Virus attachment, replication, pathogenesis, andtransmission

Much of the available data on tropism of influenza viruses in the

human respiratory tract has been derived from few autopsy studies

of fatal cases. Although highly informative to identify the cell types

that are infected in vivo and to understand pathogenesis, these data

are generally restricted to late phases of the infection. In attempts to

compensate for the lack of data in humans, the effect of receptor

specificity on tropism, pathogenesis, and transmission has also been

studied in a number of animal models like ferrets, pigs, guinea pigs,

and mice, and in primary cell lines of human and ferret airway

epithelium, and explants of ferret, pig, or human origin (reviewed

in Chan et al, 2013a). However, it should be noted that—despite the

recent increase in detailed knowledge on fine specificities of HA

interacting with different glycans in vitro (see above)—insight on

the relevance of interactions between HA and various glycans in

vivo is still largely limited to the coarse discrimination between

a2,6-SA and a2,3-SA alone.

Virus attachment

Virus histochemistry provides a method to investigate and compare

attachment patterns of influenza viruses to cells of various tissues

from different hosts, without a requirement of detailed prior

knowledge on the specific receptors. For virus histochemistry, host

tissue sections are incubated with inactivated, FITC-labeled influ-

enza viruses, much like immunohistochemistry employs FITC-

labeled antibodies. The FITC signals can be enhanced, resulting in a

bright red precipitates where the viruses (or antibodies in immuno-

histochemistry) attach. Attachment patterns of human and avian

influenza viruses in the (human) respiratory tract have been shown

to be a good indicator for virus cell and tissue tropism. Attachment

patterns of a number of avian influenza viruses and human or

mutant influenza viruses that are transmissible between humans or

ferrets are summarized in Table 2.

Broadly speaking, avian influenza viruses with a preference for

a2,3-SA were shown to attach to cells of the lower respiratory

tract, to type II pneumocytes and non-ciliated cells. In contrast,

viruses that can transmit between mammals and have a preference

for a2,6-SA were shown to attach to cells of the upper respiratory

tract, primarily to ciliated cells. These patterns of virus attachment

were in agreement with the receptor distribution in human respira-

tory tissue as determined upon staining with various lectins (see

above).

The dual receptor specificity of the 2013 H7N9 viruses was also

reflected by their attachment patterns; H7N9 viruses were shown to

attach to both cells in the upper and the lower respiratory tract of

humans, and to both type I and II pneumocytes (van Riel et al,

2013; Table 2). Some discrepancies, however, were noted in attach-

ment studies using recombinant HA proteins. Dortmans et al (2013)

primarily observed attachment in the submucosal glands of the

human upper respiratory tract, and Tharakaraman et al (2013a)

Table 2. Attachment of influenza viruses throughout the human respiratory tract as determined by virus histochemistry

Nasal turbinates Trachea Bronchus Bronchiole Alveoli

Virus SA Score Cell type Score Cell type Score Cell type Score Cell type Score Cell type

H3N2-Hu a2,6 ++ C ++ C/G ++ C/G ++ C + I

H1N1-Hu a2,6 ++ C/G ++ C/G ++ C/G + C + I

pH1N1-Hu a2,6 + C ++ C/G ++ C/G ++ C + I

H7N7-Av a2,3 +/ nd +/ nd +/ nd + N + II

H7N9-Av a2,6/a2,3 ++ C + C ++ C/G ++ C/N ++ I/II

H5N1-Av a2,3 + N + II

H5N1-Mu a2,6 ++ C/G + C/G ++ C/G ++ C ++ I

Attachment of human (Hu), avian (Av), and avian influenza with mutations (Mu) that change the receptor specificity (SA; van Riel et al, 2007, 2010, 2013;Chutinimitkul et al, 2010b). The mean abundance of cells to which virus attached was scored as follows: , no attachment; +/, attachment to rare or few cells;+, attachment to a moderate number of cells; ++, attachment to many cells. The predominant cell type to which the virus attached is indicated as follows: C,ciliated cells; N, non-ciliated cells; G, goblet cells; Ι, type Ι pneumocytes; ΙΙ, type ΙΙ pneumocytes; nd, not determined.



831

only observed limited attachment to the apical epithelial cells of the

human trachea. Although influenza viruses that bind a2,3-SA or

a2,6-SA thus display distinct attachment patterns to cells of the

human respiratory tract, small—but potentially important—differ-

ences in attachment patterns of viruses with similar receptor

specificity have also been observed. For example, H5N1 influenza

viruses with the mutations Asn-182-Lys or Gln-226-Leu and Gly-228-

Ser both displayed a preference for a2,6-SA, yet their attachment

patterns were not identical; while both viruses attached to nasal

turbinate tissue, only H5N1 with Asn-182-Lys attached to the trachea

of ferrets (Chutinimitkul et al, 2010b). The 1918 H1N1 and 1957

H2N2 pandemic influenza viruses were also both shown to bind

a2,6-SA, yet displayed differences in attachment patterns; whereas

H2N2 virus attached to the glycocalyx, goblet cells, and submucosal

glands, H1N1 virus showed predominant staining of goblet cells in

the human trachea (Jayaraman et al, 2012). The correlation between

virus attachment to cells throughout the respiratory tract and receptor

preference beyond a2,6-linkage or a2,3-linkage alone is not fully

understood and warrants further investigation.

Virus replication in primary epithelial cell cultures

Although influenza virus attachment studies have shown great

value, not all cells to which influenza viruses can bind are subse-

quently also infected (Rimmelzwaan et al, 2007), and potentially

not all infected cells produce and release infectious virus particles.

Moreover, mucus present in the respiratory tract may hamper influ-

enza virus binding and infection. Mucus from the human respira-

tory tract predominately contains a2,3-SA receptors (Couceiro et al,

1993; Lamblin & Roussel, 1993), although some a2,6-SA receptors

were also detected (Breg et al, 1987). Incubation of different influ-

enza viruses with a range of receptor specificities revealed that

particularly viruses with a2,3 specificity were inhibited by human

mucus (Couceiro et al, 1993).

Human and ferret differentiated airway epithelial cells are

frequently used to study the effect of mammalian adaptation muta-

tions on infection and replication of influenza viruses. Differentiated

airway epithelial cells form a pseudo-stratified epithelium that

closely resembles respiratory epithelium, including the presence of

cilia and secretion of mucus. In human differentiated airway epithe-

lial cells, avian influenza viruses infected ciliated cells and to a

lesser extent non-ciliated cells, whereas human influenza viruses

infected non-ciliated cells and to a lesser extent ciliated cells. This is

in contrast with virus attachment studies using human tissues with-

out culturing, which revealed binding of human influenza viruses

primarily to ciliated cells. Apparently, ciliated cells primarily

expressed a2,3-SA and non-ciliated cells expressed a2,6-SA upon

culturing (Matrosovich et al, 2004; Thompson et al, 2006; Wan &

Perez, 2007), although the presence of a2,6-SA on ciliated cells has

been reported in some studies (Ibricevic et al, 2006; Schrauwen

et al, 2013).

In contrast to human differentiated airway epithelial cells, cili-

ated differentiated airway epithelial cells from ferrets were shown

to express a2,6-SA, while non-ciliated cells expressed a2,3-SA.Both human and avian influenza viruses replicated efficiently in

differentiated airway epithelial cells from ferrets, but avian viruses

had a lower initial infection rate. Using electron microscopy, it was

shown that human influenza viruses were predominantly

released from the ciliated cells, and release of avian viruses was

only sporadically detected and only from non-ciliated cells, in

agreement with the distribution of a2,3-SA and a2,6-SA (Zeng

et al, 2013).

Although differentiated airway epithelial cell cultures are quite

valuable for various studies, it should thus be noted that cultures of

human and ferret origin may behave differently and that receptor

expression upon culture may not be representative for the in vivo

human respiratory tract tissues.

Virus replication in animal models

Unlike seasonal human influenza viruses, H5N1 viruses generally

do not attach to the upper respiratory tract of ferrets or humans

(van Riel et al, 2006, 2010; Chutinimitkul et al, 2010b). However, in

contrast to the results from attachment studies, H5N1 virus was

reported to replicate both in human upper respiratory tract tissue ex

vivo (Nicholls et al, 2007b) and in the ferret upper respiratory tract

in vivo (Maines et al, 2005, 2006). It is possible that the high virus

titers found in the ferret upper respiratory tract are explained in part

by virus replication in the olfactory epithelium, which is related to

the nervous system and lines a large part of the nasal cavity

(Schrauwen et al, 2012). It has also been reported that H5N1

influenza viruses with mutations that change receptor specificity

from a2,3-SA to a2,6-SA did not replicate more efficiently in the

upper respiratory tract of ferrets than their wild-type counterparts

(Wang et al, 2010; Maines et al, 2011; Herfst et al, 2012). This may

be explained by a requirement for additional adaptive mutations in

HA to accommodate the mutations responsible for a2,6-SA binding,

but more research is needed to identify such “fitness-enhancing”

mutations.

The dual receptor specificity of the H7N9 virus (noted above)

was reflected in the attachment patterns to the upper and lower

respiratory tract of humans (van Riel et al, 2013). In agreement with

receptor specificity and patterns of virus attachment, the H7N9 virus

was shown to replicate in both trachea and lung explants (with

higher titers in the lung explants) and in human lung cell cultures

(Belser et al, 2013a; Knepper et al, 2013; Zhou et al, 2013). In

ferrets, the H7N9 virus replicated both in the upper and lower

respiratory tract (Kreijtz et al, 2013; Zhang et al, 2013a; Zhu et al,

2013).

Receptor specificity and pathogenesis

The site of influenza virus replication is potentially one of the most

critical determinants of pathogenesis. H5N1 viruses were shown to

replicate efficiently in the lower respiratory tract in type II pneu-

mocytes (Gu et al, 2007), probably related to the specificity for

a2,3-SA, and contributing to pathogenicity. For pH1N1 virus,

amino acid substitution Asp-225-Gly that resulted in increased

binding to a2,3-SA was also linked to enhanced disease

(Chutinimitkul et al, 2010a; Kilander et al, 2010; Watanabe et al,

2011). Potentially, the dual receptor specificity of the H7N9 virus

could be responsible for the severe disease and high mortality

observed in humans. Indeed, in various mammalian model

systems, the H7N9 virus was shown to be more pathogenic than

typical human influenza viruses, which was probably related to

efficient virus replication throughout the respiratory tract (Belser

et al, 2013a; Kreijtz et al, 2013; Watanabe et al, 2013). However,

it should be noted that not all avian influenza viruses (with

specificity for a2,3-SA) are pathogenic in humans; human



832

volunteers that were infected with various avian influenza viruses

only displayed mild symptoms (Beare & Webster, 1991), thus

emphasizing that other viral (e.g., NS1, polymerase, NA) and host

factors (e.g., adaptive and innate immune responses) also critically

determine pathogenesis.

Virus transmissionIt has been suggested that (i) inefficient virus attachment to (upper)

respiratory tissues, (ii) virus replication at only low levels in these

tissues, and (iii) poor virus release and aerosolization of virus parti-

cles may provide an explanation why influenza viruses with

a2,3-SA preference are not transmitted via the airborne route

between mammals (Sorrell et al, 2011). In addition, it was proposed

that for a virus that requires a2,3-SA receptors, a higher dose is

needed to infect a new mammalian host, which makes it less likely

to transmit (Roberts et al, 2011). Tumpey et al reported that 1918 H1N1

influenza viruses with a2,3-SA receptor specificity were not trans-

mitted via the airborne route between ferrets, while viruses with

dual receptor specificity were transmitted, albeit less efficiently as

compared to viruses with a2,6-SA receptor specificity. Based on

these results, it was postulated that a loss of binding to a2,3-SA is

important for airborne transmission (Tumpey et al, 2007). While a

loss of binding to a2,3-SA can result in more efficient airborne trans-

mission, it should be noted that early H2N2 and H3N2 pandemic

viruses (that spread efficiently between humans) still had dual

receptor specificity (Matrosovich et al, 2000). Both the airborne

transmissible H5N1 viruses and the 2013 H7N9 virus displayed dual

receptor specificity and were transmitted between ferrets, but for

these viruses transmission was less efficient compared to pandemic

and seasonal human influenza viruses (Chen et al, 2012; Herfst

et al, 2012; Imai et al, 2012; Belser et al, 2013a; Richard et al, 2013;

Watanabe et al, 2013; Xu et al, 2013; Zhang et al, 2013a,d; Zhu

et al, 2013). The current common belief is that for airborne trans-

mission between humans or ferrets, influenza viruses require

human over avian receptor binding preference; viruses with exclu-

sive a2,3-SA binding are generally not transmitted, viruses with

dual receptor specificity are often transmitted inefficiently, while

influenza viruses with full-blown airborne transmission between

ferrets or humans generally had a strong a2,6-SA binding prefer-

ence. It is important to identify whether a fine specificity in receptor

binding beyond a2,3-SA/a2,6-SA is required for airborne influenza

virus transmission between mammals.

Determinants of airborne transmission beyond receptorspecificity

It is clear from airborne transmission studies on pandemic influenza

and avian H5N1, H9N2, and H7N9 influenza viruses that a change

of receptor specificity is a necessity, but by itself is not enough to

result in airborne transmission between mammals. But what are

other adaptations of the HA or other viral proteins required for

airborne transmission between mammals?

HA stabilityUpon virus attachment to SA receptors on the cell surface and inter-

nalization into endosomes, a low-pH-triggered conformational change

in HA mediates fusion of the viral and endosomal membranes to

release the virus genome in the cytoplasm. The switch of influenza

virus HA from a metastable non-fusogenic to a stable fusogenic

conformation can also be triggered at neutral pH when the HA is

exposed to increasing temperature. This conformational change in

HA is biochemically indistinguishable from the change triggered by

low pH (Haywood & Boyer, 1986; Carr et al, 1997).

Generally, the threshold pH of fusion for HAs of human

influenza viruses is lower than that of avian influenza isolates

(Galloway et al, 2013). Possibly, the optimal fusion pH of a virus

depends on the original host. For avian influenza viruses that repli-

cate in the intestinal tract of birds and are transmitted via surface

waters, fusion at a high pH might be more optimal. In contrast, effi-

cient transmission through the air to and from the relatively acidic

human nasal cavity as site of replication could require a difference

in pH threshold (England et al, 1999; Washington et al, 2000).

Lowering the optimal fusion pH for viruses with an avian H5 HA

resulted in enhanced replication in the ferret upper respiratory tract

and for some viruses in more efficient contact transmission (Shelton

et al, 2013; Zaraket et al, 2013). The HA of the airborne transmissi-

ble H5N1 virus described by Imai et al (2012) contained the substi-

tution Thr-318-Ile, which is located proximal to the fusion peptide.

The wild-type H5N1 virus (VN1203) had a pH 5.7 fusion threshold

and introduction of mutations that change receptor specificity

increased the fusion threshold to pH 5.9. However, subsequent intro-

duction of Thr-318-Ile reduced the threshold of fusion to pH 5.5. In

agreement with this increased pH stability, Thr-318-Ile also

increased the thermostability of this HA (Imai et al, 2012). Structural

studies revealed that the Thr-318-Ile mutation stabilized the fusion

peptide within the HA monomer (Xiong et al, 2013a). Notably,

substitution His-110-Tyr in a recombinant protein based on the

airborne transmissible virus described by Herfst et al also appeared

to increase the stability of the HA at high temperatures (de Vries

et al, 2014). His-110-Tyr is located at the trimer interface and forms

a hydrogen bond with 413-Asn of the adjacent monomer, thereby

stabilizing the trimeric HA protein (Zhang et al, 2013b). Whether

this substitution has the same effect on pH stability is still unknown.

The 2013 H7N9 viruses that showed limited airborne transmis-

sion between ferrets were shown to have a relatively unstable HA,

with conformational changes induced already at relatively low

temperatures and a threshold pH for membrane fusion at pH 5.6–5.8

(Richard et al, 2013). Given the observed differences in threshold

pH of fusion and thermostability between HAs of human and avian

influenza viruses, and the acquired stability mutations in HA of

airborne H5N1 viruses, HA stability as an adaptation marker of

human influenza viruses clearly warrants further study. It is impor-

tant to note that the observed changes in pH stability and thermosta-

bility may merely be surrogate markers for other stability

phenotypes, for example stability in aerosols, in mucus, or upon

exposure to air.

HA-NA balanceAs a consequence of the switch in HA receptor binding preference

when avian influenza viruses adapt to mammals, subsequent adap-

tation of NA may be required to maintain an optimal balance

between attachment to SA and cleavage of SA. Low NA activity

can result in inefficient SA cleavage and subsequent aggregation of

virus particles, which can result in low infectious virus titers

despite high genome copy numbers (Palese et al, 1974; Liu et al,



833

1995; de Wit et al, 2010). In contrast, low binding affinity of HA to

receptors in combination with high NA activity would likely disturb

efficient binding to the host cells. Avian influenza virus NAs specif-

ically cleave a2,3-SA, whereas human influenza virus NAs cleave

both a2,3-SA and a2,6-SA. It is thus likely that the NA of avian

viruses needs to co-adapt to the receptor specificity of the HA, for

efficient virus attachment and entry, virus release, and potentially

transmission (Baum & Paulson, 1991; Kobasa et al, 1999). In this

light, it is important to note that several airborne transmissible H5

viruses contained NA derived from human influenza viruses (Chen

et al, 2012; Imai et al, 2012; Zhang et al, 2013d), but that the fully

avian H5N1 viruses described by Herfst et al did not require

any changes in the NA gene for airborne transmission (Herfst

et al, 2012).

Polymerase proteinsIn addition to the HA and NA proteins, the influenza virus

polymerase proteins were shown to require adaptation for efficient

replication to occur upon transmission from animals to humans

(reviewed in Manz et al, 2013). Herfst et al introduced the well-

known adaptive mutation Gly-627-Lys in the PB2 gene of an H5N1

virus that subsequently accumulated numerous additional substitu-

tions upon ferret passage in polymerase proteins and nucleoprotein

to yield airborne transmissible virus. The Lys at position 627 of

PB2 was previously described to contribute to successful replica-

tion in mammalian cells at lower temperature (Almond, 1977;

Subbarao et al, 1993; Hatta et al, 2001; Massin et al, 2001; Yamad-

a et al, 2010), and airborne transmission between mammals (Steel

et al, 2009; Van Hoeven et al, 2009). Efficient replication at lower

temperature is thought to be important for adaptation to humans,

since the temperature in the human upper respiratory tract is

approximately 33°C, which is much lower compared to the

temperature in the avian intestinal tract (~41°C), the site of

replication of avian viruses.

Amino acid substitutions at positions 590/591 and at position

701 of PB2 were also shown to result in more efficient replication in

mammalian cells (Steel et al, 2009; Yamada et al, 2010). Of the

H7N9 virus genome sequences available from public databases,

65% contained 627-Lys, 9% contained 701-Asn, and 8% contained

591-Lys, in contrast to avian H7N9 viruses that retained 627-Glu,

701-Asp, and 591-Gln (Chen et al, 2013; Gao et al, 2013b). So far,

adaptive changes in PB1, PA, and NP that may contribute to virus

adaptation in mammals and airborne transmission have not been

investigated in detail, but are urgently warranted.

Challenges and future outlook

Recent work on virus attachment, replication, pathogenesis, and

transmission and follow-up work on structural determinants of HA

receptor specificity and stability have shed important new light on

influenza virus adaptation to mammalian hosts. Imai et al and

Herfst et al described different amino acid substitutions in H5N1

virus HA that represented remarkably similar phenotypic traits lead-

ing to airborne virus transmission. Some of these substitutions were

also noted in 2013 H7N9 viruses with limited airborne transmissibil-

ity, in an airborne H9N2 virus, and in the pandemic viruses of the

last century. Some of the substitutions of the laboratory-derived

airborne H5N1 viruses and combinations of such substitutions were

already detected in naturally circulating H5N1 viruses (Russell et al,

2012). However, it should be noted that these mutations do not

necessarily result in the same phenotype in all H5N1 strains or in

strains of other influenza virus subtypes. Moreover, it is possible

that there are evolutionary constraints to the accumulation of

genetic changes required to yield an airborne transmission pheno-

type, making the emergence of such viruses possibly an unlikely

event (Russell et al, 2012). Further identification of the genetic and

phenotypic changes required for host adaptation and transmission

of influenza viruses remains an important research topic to facilitate

risk assessment for the emergence of zoonotic and pandemic

influenza.

In currently circulating strains of clade 2.2.1 H5N1 containing a

deletion in the 130-loop and lacking a glycosylation site at position

160, the substitutions 224-Asn and 226-Leu resulted in a switch of

receptor specificity (Tharakaraman et al, 2013b). We know that

there are many more ways by which H5N1 viruses can adapt to

attach to a2,6-SA. Some natural human H5N1 virus isolates bind

to both a2,3-SA and a2,6-SA due to substitutions in and around

the RBS (Yamada et al, 2006; Crusat et al, 2013). In addition,

mutational analyses of H5 HA based on analogies with other influ-

enza virus subtypes identified several substitutions that caused a

switch in receptor specificity and virus attachment to upper respi-

ratory tract tissues of humans. Although none of these human

virus isolates and mutant viruses were capable of airborne trans-

mission between mammals (Maines et al, 2006; Stevens et al,

2006b, 2008; Chutinimitkul et al, 2010b; Paulson & de Vries,

2013), it is clear that there are multiple ways by which circulating

H5N1 strains can change receptor specificity and adapt to replica-

tion in mammals.

Yang et al (2013) have shown that introduction of substitution

228-Ser in H7N9 HA that already had 226-Leu resulted in overall

increased binding to a2,3-SA and a2,6-SA, but not in a switch in

receptor preference. Based on structural studies, it was anticipated

that Gly-228-Ser would optimize interactions with both a2,3-SAand a2,6-SA. Gly-228-Ser resulted in increased binding of the H7N9

HA to the apical surface of tracheal and alveolar tissues of humans

(Tharakaraman et al, 2013a). At present, it is unclear whether

other amino acid substitutions would further fine-tune the receptor

binding preference of the H7N9 viruses and could potentially

increase the airborne transmissibility under laboratory or natural

conditions.

Although our knowledge of receptor specificity and its implica-

tions for transmission and pathogenesis has improved since the

initial characterization of the influenza virus RBS, there are still

many unknowns. The lack of knowledge of which cells are the

primary target of airborne transmissible viruses and the type and

distribution of receptors they express hampers the interpretation of

receptor binding data and structural studies. Major current interest

is focused on receptor binding assays such as glycan arrays, solid-

phase binding assays, and agglutination assays using modified

erythrocytes. But without knowledge on which receptors are rele-

vant for influenza virus attachment, replication, and transmission

in vivo, it is impossible to assess which binding assays best repre-

sent influenza virus attachment in the respiratory tract. As a

consequence, studies should focus more on identification of the

relevant functional glycans in humans and animal models. The



834

possibility that a plethora of receptors in the human respiratory

tract can be utilized for the attachment of influenza viruses and

are relevant for replication and transmission should be considered.

Based on studies of the glycome of human and animal hosts, it is

clear that a large number of glycan structures are not represented

on glycan arrays (Walther et al, 2013). A potential way forward

would be the use of shotgun glycan arrays that employ receptors

of relevant tissues and cells of the human respiratory tract rather

than random synthetic glycans (Yu et al, 2012). At present, such

studies are limited since they generally employ all glycans

expressed in the respiratory tract rather than just those receptors

expressed on relevant cell types at the apical surface of the respira-

tory tract that are accessible upon influenza virus infection. Future

work should clearly focus on improvement of the methods avail-

able in glycobiology. Similarly, X-ray crystal structures of human

and avian receptor analogs in complex with HAs of human and

influenza viruses have revealed crucial structural determinants for

binding to a2,3-SA and a2,6-SA receptor analogs. But potentially

these structural studies could also be complemented with influenza

HAs in complex with a variety of sialylated glycan structures,

to increase understanding of the structural determinants of HA

receptor binding.

There is a clear need for phenotypic assays to predict which

animal influenza viruses can infect humans and transmit between

humans. One major unknown is to what extent the transmissibility

of influenza viruses in ferret and guinea pig transmission models

can be extrapolated to humans. Although thus far there has been

good correspondence between the transmissibility of avian and

human influenza viruses between humans and the ferret transmis-

sion model, it is not certain that all viruses that transmit between

ferrets will also transmit between humans. It is also not clear how

the ferret and guinea pig models compare with respect to airborne

transmission of animal influenza viruses. Better understanding

about the behavior of airborne transmissible viruses in vitro may

allow replacement of some animal models with in vitro methods

and contribute to reduction, replacement, and refinement in animal

experiments (Russell & Burch, 1959; Belser et al, 2013b). In addi-

tion, such phenotypical assays may complement ongoing surveil-

lance studies to identify biological traits of circulating influenza

viruses that could trigger immediate action or attention. So far,

when data obtained with attachment studies, infection studies using

primary epithelial cell lines explants, and animal studies were

compared, the correspondence was not always obvious, and there-

fore, more work in this field is also needed. While it may never be

feasible to perfectly predict the adaptation of animal influenza

viruses to humans, increased knowledge from animal studies, in

vitro assays, and structural analyses collectively will almost

certainly improve such predictions.

AcknowledgementsThe authors are supported by EU FP7 programs ANTIGONE and EMPERIE,

NIAID/NIH contract HHSN266200700010C, and an Intra European Marie

Curie Fellowship (PIEF-GA-2009-237505). We thank C.H. Hokke, B. Mänz, D.F.

Burke, E.J.A. Schrauwen, D. van Riel, E. de Vries, C.A. de Haan, and S. Herfst

for critically reading the manuscript and their helpful comments.

Author contributionsMG and RAMF wrote the manuscript.

Conflict of interestThe authors declare that they have no conflict of interest.

References

Ablan S, Rawat SS, Blumenthal R, Puri A (2001) Entry of influenza virus into

a glycosphingolipid-deficient mouse skin fibroblast cell line. Arch Virol 146:

2227 – 2238

Aich U, Beckley N, Shriver Z, Raman R, Viswanathan K, Hobbie S, Sasisekharan R

(2011) Glycomics-based analysis of chicken red blood cells provides insight

into the selectivity of the viral agglutination assay. FEBS J 278: 1699 – 1712

Almond JW (1977) A single gene determines the host range of influenza virus.

Nature 270: 617 – 618

Bateman AC, Karamanska R, Busch MG, Dell A, Olsen CW, Haslam SM (2010)

Glycan analysis and influenza A virus infection of primary swine

respiratory epithelial cells: the importance of NeuAcalpha2-6 glycans.

J Biol Chem 285: 34016 – 34026

Baum LG, Paulson JC (1991) The N2 neuraminidase of human influenza virus

has acquired a substrate specificity complementary to the hemagglutinin

receptor specificity. Virology 180: 10 – 15

Beare AS, Webster RG (1991) Replication of avian influenza viruses in

humans. Arch Virol 119: 37 – 42

Belser JA, Blixt O, Chen LM, Pappas C, Maines TR, Van Hoeven N, Donis R,

Busch J, McBride R, Paulson JC, Katz JM, Tumpey TM (2008) Contemporary

North American influenza H7 viruses possess human receptor specificity:

Implications for virus transmissibility. Proc Natl Acad Sci USA 105:

7558 – 7563

Belser JA, Gustin KM, Pearce MB, Maines TR, Zeng H, Pappas C, Sun X, Carney

PJ, Villanueva JM, Stevens J, Katz JM, Tumpey TM (2013a) Pathogenesis

and transmission of avian influenza A (H7N9) virus in ferrets and mice.

Nature 501: 556 – 559

Belser JA, Maines TR, Katz JM, Tumpey TM (2013b) Considerations regarding

appropriate sample size for conducting ferret transmission experiments.

Future Microbiol 8: 961 – 965

Breg J, Van Halbeek H, Vliegenthart JF, Lamblin G, Houvenaghel MC, Roussel

P (1987) Structure of sialyl-oligosaccharides isolated from bronchial mucus

glycoproteins of patients (blood group O) suffering from cystic fibrosis. Eur

J Biochem 168: 57 – 68

Brockhausen I, Schachter H, Stanley P (2009) O-GalNAc glycans. In Essentials

of Glycobiology, Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P,

Bertozzi CR, Hart GW, Etzler ME (eds), 2nd edn, Chapter 9. Cold Spring

Harbor, NY: Cold Spring Harbor Laboratory Press

Carr CM, Chaudhry C, Kim PS (1997) Influenza hemagglutinin is

spring-loaded by a metastable native conformation. Proc Natl Acad Sci

USA 94: 14306 – 14313

Centers for Disease Control and Prevention (2004) Update: influenza activity–

United States, 2003–04 season. MMWR Morb Mortal Wkly Rep 53:

284 – 287

Chan RW, Chan MC, Nicholls JM, Malik Peiris JS (2013a) Use of ex vivo and in

vitro cultures of the human respiratory tract to study the tropism and

host responses of highly pathogenic avian influenza A (H5N1) and other

influenza viruses. Virus Res 178: 133 – 145

Chan RW, Karamanska R, Van Poucke S, Van Reeth K, Chan IW, Chan MC,

Dell A, Peiris JS, Haslam SM, Guan Y, Nicholls JM (2013b) Infection

of swine ex vivo tissues with avian viruses including H7N9 and

correlation with glycomic analysis. Influenza Other Respir Viruses 7:

1269 – 1282



835

Chen LM, Blixt O, Stevens J, Lipatov AS, Davis CT, Collins BE, Cox NJ, Paulson

JC, Donis RO (2012) In vitro evolution of H5N1 avian influenza virus

toward human-type receptor specificity. Virology 422: 105 – 113

Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, Yao H, Wo J, Fang Q, Cui D,

Li Y, Yao X, Zhang Y, Wu H, Zheng S, Diao H, Xia S, Chan KH, Tsoi HW,

Teng JL et al (2013) Human infections with the emerging avian influenza

A H7N9 virus from wet market poultry: clinical analysis and

characterisation of viral genome. Lancet 381: 1916 – 1925

Childs RA, Palma AS, Wharton S, Matrosovich T, Liu Y, Chai W,

Campanero-Rhodes MA, Zhang Y, Eickmann M, Kiso M, Hay A,

Matrosovich M, Feizi T (2009) Receptor-binding specificity of pandemic

influenza A (H1N1) 2009 virus determined by carbohydrate microarray.

Nat Biotechnol 27: 797 – 799

Chou HH, Takematsu H, Diaz S, Iber J, Nickerson E, Wright KL, Muchmore EA,

Nelson DL, Warren ST, Varki A (1998) A mutation in human CMP-sialic

acid hydroxylase occurred after the Homo-Pan divergence. Proc Natl Acad

Sci USA 95: 11751 – 11756

Chu VC, Whittaker GR (2004) Influenza virus entry and infection require host

cell N-linked glycoprotein. Proc Natl Acad Sci USA 101: 18153 – 18158

Chutinimitkul S, Herfst S, Steel J, Lowen AC, Ye J, van Riel D, Schrauwen EJ,

Bestebroer TM, Koel B, Burke DF, Sutherland-Cash KH, Whittleston CS,

Russell CA, Wales DJ, Smith DJ, Jonges M, Meijer A, Koopmans M,

Rimmelzwaan GF, Kuiken T et al (2010a) Virulence-associated substitution

D222G in the hemagglutinin of 2009 pandemic influenza A(H1N1) virus

affects receptor binding. J Virol 84: 11802 – 11813

Chutinimitkul S, van Riel D, Munster VJ, van den Brand JM, Rimmelzwaan GF,

Kuiken T, Osterhaus AD, Fouchier RA, de Wit E (2010b) In vitro assessment

of attachment pattern and replication efficiency of H5N1 influenza A

viruses with altered receptor specificity. J Virol 84: 6825 – 6833

Connor RJ, Kawaoka Y, Webster RG, Paulson JC (1994) Receptor specificity in

human, avian, and equine H2 and H3 influenza virus isolates. Virology

205: 17 – 23

Costa T, Chaves AJ, Valle R, Darji A, van Riel D, Kuiken T, Majo N, Ramis A

(2012) Distribution patterns of influenza virus receptors and viral

attachment patterns in the respiratory and intestinal tracts of seven avian

species. Vet Res 43: 28

Couceiro JN, Paulson JC, Baum LG (1993) Influenza virus strains selectively

recognize sialyloligosaccharides on human respiratory epithelium; the role

of the host cell in selection of hemagglutinin receptor specificity. Virus Res

29: 155 – 165

Crusat M, Liu J, Palma AS, Childs RA, Liu Y, Wharton SA, Lin YP, Coombs PJ,

Martin SR, Matrosovich M, Chen Z, Stevens DJ, Hien VM, Thanh TT, le Nhu

NT, Nguyet LA, Ha do Q, van Doorn HR, Hien TT, Conradt HS et al (2013)

Changes in the hemagglutinin of H5N1 viruses during human infection–

influence on receptor binding. Virology 447: 326 – 337

Dortmans JC, Dekkers J, Wickramasinghe IN, Verheije MH, Rottier PJ, van

Kuppeveld FJ, de Vries E, de Haan CA (2013) Adaptation of novel H7N9

influenza A virus to human receptors. Sci Rep 3: 3058

England RJ, Homer JJ, Knight LC, Ell SR (1999) Nasal pH measurement: a

reliable and repeatable parameter. Clin Otolaryngol Allied Sci 24:

67 – 68

Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D,

Rimmelzwaan GF, Olsen B, Osterhaus AD (2005) Characterization of a

novel influenza A virus hemagglutinin subtype (H16) obtained from

black-headed gulls. J Virol 79: 2814 – 2822

Fouchier RA, Schneeberger PM, Rozendaal FW, Broekman JM, Kemink SA,

Munster V, Kuiken T, Rimmelzwaan GF, Schutten M, Van Doornum GJ, Koch

G, Bosman A, Koopmans M, Osterhaus AD (2004) Avian influenza A virus

(H7N7) associated with human conjunctivitis and a fatal case of acute

respiratory distress syndrome. Proc Natl Acad Sci USA 101: 1356 – 1361

Franca M, Stallknecht DE, Howerth EW (2013) Expression and distribution

of sialic acid influenza virus receptors in wild birds. Avian Pathol 42:

60 – 71

Galloway SE, Reed ML, Russell CJ, Steinhauer DA (2013) Influenza HA

subtypes demonstrate divergent phenotypes for cleavage activation and

pH of fusion: implications for host range and adaptation. PLoS Pathog 9:

e1003151

Gambaryan A, Yamnikova S, Lvov D, Tuzikov A, Chinarev A, Pazynina G,

Webster R, Matrosovich M, Bovin N (2005) Receptor specificity

of influenza viruses from birds and mammals: new data on

involvement of the inner fragments of the carbohydrate chain. Virology

334: 276 – 283

Gambaryan AS, Matrosovich TY, Philipp J, Munster VJ, Fouchier RA, Cattoli G,

Capua I, Krauss SL, Webster RG, Banks J, Bovin NV, Klenk HD, Matrosovich

MN (2012) Receptor-binding profiles of H7 subtype influenza viruses in

different host species. J Virol 86: 4370 – 4379

Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS,

Bovin NV, Matrosovich MN (1997) Specification of receptor-binding

phenotypes of influenza virus isolates from different hosts using synthetic

sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and

influenza B viruses share a common high binding affinity for 60-sialyl(N-acetyllactosamine). Virology 232: 345 – 350

Gamblin SJ, Haire LF, Russell RJ, Stevens DJ, Xiao B, Ha Y, Vasisht N,

Steinhauer DA, Daniels RS, Elliot A, Wiley DC, Skehel JJ (2004) The

structure and receptor binding properties of the 1918 influenza

hemagglutinin. Science 303: 1838 – 1842

Gamblin SJ, Skehel JJ (2010) Influenza hemagglutinin and neuraminidase

membrane glycoproteins. J Biol Chem 285: 28403 – 28409

Gao HN, Lu HZ, Cao B, Du B, Shang H, Gan JH, Lu SH, Yang YD, Fang Q, Shen

YZ, Xi XM, Gu Q, Zhou XM, Qu HP, Yan Z, Li FM, Zhao W, Gao ZC, Wang

GF, Ruan LX et al (2013a) Clinical findings in 111 cases of influenza A

(H7N9) virus infection. N Engl J Med 368: 2277 – 2285

Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X,

Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y et al

(2013b) Human infection with a novel avian-origin influenza A (H7N9)

virus. N Engl J Med 368: 1888 – 1897

Geisler C, Jarvis DL (2011) Effective glycoanalysis with Maackia amurensis

lectins requires a clear understanding of their binding specificities.

Glycobiology 21: 988 – 993

Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, Basler

CF, Taubenberger JK, Palese P (2005) A single amino acid substitution in

1918 influenza virus hemagglutinin changes receptor binding specificity.

J Virol 79: 11533 – 11536

Gottschalk A (1957) Neuraminidase: the specific enzyme of influenza virus

and Vibrio cholerae. Biochim Biophys Acta 23: 645 – 646

Gu J, Xie Z, Gao Z, Liu J, Korteweg C, Ye J, Lau LT, Lu J, Zhang B, McNutt MA,

Lu M, Anderson VM, Gong E, Yu AC, Lipkin WI (2007) H5N1 infection of

the respiratory tract and beyond: a molecular pathology study. Lancet 370:

1137 – 1145

Guan Y, Shortridge KF, Krauss S, Webster RG (1999) Molecular

characterization of H9N2 influenza viruses: were they the donors of the

“internal” genes of H5N1 viruses in Hong Kong? Proc Natl Acad Sci USA 96:

9363 – 9367

Ha Y, Stevens DJ, Skehel JJ, Wiley DC (2001) X-ray structures of H5 avian and

H9 swine influenza virus hemagglutinins bound to avian and human

receptor analogs. Proc Natl Acad Sci USA 98: 11181 – 11186



836

Hart GW (2013) Thematic minireview series on glycobiology and extracellular

matrices: glycan functions pervade biology at all levels. J Biol Chem 288:

6903

Hatta M, Gao P, Halfmann P, Kawaoka Y (2001) Molecular basis for high

virulence of Hong Kong H5N1 influenza A viruses. Science 293: 1840 – 1842

Haywood AM, Boyer BP (1986) Time and temperature dependence of influenza

virus membrane fusion at neutral pH. J Gen Virol 67(Pt 12): 2813 – 2817

Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ,

Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF,

Osterhaus AD, Fouchier RA (2012) Airborne transmission of influenza

A/H5N1 virus between ferrets. Science 336: 1534 – 1541

Hirst GK (1941) The agglutination of red cells by allantoic fluid of chick

embryos infected with influenza virus. Science 94: 22 – 23

Ibricevic A, Pekosz A, Walter MJ, Newby C, Battaile JT, Brown EG, Holtzman

MJ, Brody SL (2006) Influenza virus receptor specificity and cell tropism in

mouse and human airway epithelial cells. J Virol 80: 7469 – 7480

Ichikawa S, Nakajo N, Sakiyama H, Hirabayashi Y (1994) A mouse B16

melanoma mutant deficient in glycolipids. Proc Natl Acad Sci USA 91:

2703 – 2707

Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson

A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y,

Maher EA, Neumann G, Kawaoka Y (2012) Experimental adaptation of an

influenza H5 HA confers respiratory droplet transmission to a reassortant

H5 HA/H1N1 virus in ferrets. Nature 486: 420 – 428

Ito T, Couceiro JN, Kelm S, Baum LG, Krauss S, Castrucci MR, Donatelli I, Kida

H, Paulson JC, Webster RG, Kawaoka Y (1998) Molecular basis for the

generation in pigs of influenza A viruses with pandemic potential. J Virol

72: 7367 – 7373

Jayaraman A, Chandrasekaran A, Viswanathan K, Raman R, Fox JG, Sasisekharan

R (2012) Decoding the distribution of glycan receptors for human-adapted

influenza A viruses in ferret respiratory tract. PLoS One 7: e27517

Kandun IN, Wibisono H, Sedyaningsih ER, Yusharmen Hadisoedarsuno W,

Purba W, Santoso H, Septiawati C, Tresnaningsih E, Heriyanto B, Yuwono

D, Harun S, Soeroso S, Giriputra S, Blair PJ, Jeremijenko A, Kosasih H,

Putnam SD, Samaan G, Silitonga M, Chan KH et al (2006) Three

Indonesian clusters of H5N1 virus infection in 2005. N Engl J Med 355:

2186 – 2194

Kawaoka Y, Krauss S, Webster RG (1989) Avian-to-human transmission of the

PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol

63: 4603 – 4608

Kilander A, Rykkvin R, Dudman SG, Hungnes O (2010) Observed association

between the HA1 mutation D222G in the 2009 pandemic influenza A

(H1N1) virus and severe clinical outcome, Norway 2009–2010. Euro

Surveill 15: pii: 19498

Kirkeby S, Martel CJ, Aasted B (2009) Infection with human H1N1 influenza

virus affects the expression of sialic acids of metaplastic mucous cells in

the ferret airways. Virus Res 144: 225 – 232

Knepper J, Schierhorn KL, Becher A, Budt M, Tonnies M, Bauer TT, Schneider

P, Neudecker J, Ruckert JC, Gruber AD, Suttorp N, Schweiger B, Hippenstiel

S, Hocke AC, Wolff T (2013) The novel human influenza A(H7N9) virus is

naturally adapted to efficient growth in human lung tissue. MBio 4:

e00601 – 00613

Kobasa D, Kodihalli S, Luo M, Castrucci MR, Donatelli I, Suzuki Y, Suzuki T,

Kawaoka Y (1999) Amino acid residues contributing to the substrate

specificity of the influenza A virus neuraminidase. J Virol 73: 6743 – 6751

Kreijtz JH, Kroeze EJ, Stittelaar KJ, de Waal L, van Amerongen G, van Trierum

S, van Run P, Bestebroer T, Kuiken T, Fouchier RA, Rimmelzwaan GF,

Osterhaus AD (2013) Low pathogenic avian influenza A(H7N9) virus causes

high mortality in ferrets upon intratracheal challenge: a model to study

intervention strategies. Vaccine 31: 4995 – 4999

Kuchipudi SV, Nelli R, White GA, Bain M, Chang KC, Dunham S (2009)

Differences in influenza virus receptors in chickens and ducks:

implications for interspecies transmission. J Mol Genet Med 3: 143 – 151

Lam TT, Wang J, Shen Y, Zhou B, Duan L, Cheung CL, Ma C, Lycett SJ, Leung CY,

Chen X, Li L, Hong W, Chai Y, Zhou L, Liang H, Ou Z, Liu Y, Farooqui A, Kelvin

DJ, Poon LL et al (2013) The genesis and source of the H7N9 influenza

viruses causing human infections in China. Nature 502: 241 – 244

Lamblin G, Roussel P (1993) Airway mucins and their role in defence against

micro-organisms. Respir Med 87: 421 – 426

Leigh MW, Connor RJ, Kelm S, Baum LG, Paulson JC (1995) Receptor

specificity of influenza virus influences severity of illness in ferrets. Vaccine

13: 1468 – 1473

Lin YP, Xiong X, Wharton SA, Martin SR, Coombs PJ, Vachieri SG,

Christodoulou E, Walker PA, Liu J, Skehel JJ, Gamblin SJ, Hay AJ, Daniels RS,

McCauley JW (2012) Evolution of the receptor binding properties of the

influenza A(H3N2) hemagglutinin. Proc Natl Acad Sci USA 109:

21474 – 21479

Liu C, Eichelberger MC, Compans RW, Air GM (1995) Influenza type A virus

neuraminidase does not play a role in viral entry, replication, assembly, or

budding. J Virol 69: 1099 – 1106

Liu J, Stevens DJ, Haire LF, Walker PA, Coombs PJ, Russell RJ, Gamblin SJ,

Skehel JJ (2009) Structures of receptor complexes formed by

hemagglutinins from the Asian Influenza pandemic of 1957. Proc Natl

Acad Sci USA 106: 17175 – 17180

Lowen AC, Mubareka S, Tumpey TM, Garcia-Sastre A, Palese P (2006) The

guinea pig as a transmission model for human influenza viruses. Proc Natl

Acad Sci USA 103: 9988 – 9992

Lu X, Shi Y, Zhang W, Zhang Y, Qi J, Gao GF (2013) Structure and

receptor-binding properties of an airborne transmissible avian influenza A

virus hemagglutinin H5 (VN1203mut). Protein Cell 4: 502 – 511

Maines TR, Chen LM, Matsuoka Y, Chen H, Rowe T, Ortin J, Falcon A, Nguyen

TH, le Mai Q, Sedyaningsih ER, Harun S, Tumpey TM, Donis RO, Cox NJ,

Subbarao K, Katz JM (2006) Lack of transmission of H5N1 avian-human

reassortant influenza viruses in a ferret model. Proc Natl Acad Sci USA 103:

12121 – 12126

Maines TR, Chen LM, Van Hoeven N, Tumpey TM, Blixt O, Belser JA, Gustin

KM, Pearce MB, Pappas C, Stevens J, Cox NJ, Paulson JC, Raman R,

Sasisekharan R, Katz JM, Donis RO (2011) Effect of receptor binding

domain mutations on receptor binding and transmissibility of avian

influenza H5N1 viruses. Virology 413: 139 – 147

Maines TR, Lu XH, Erb SM, Edwards L, Guarner J, Greer PW, Nguyen DC,

Szretter KJ, Chen LM, Thawatsupha P, Chittaganpitch M, Waicharoen S,

Nguyen DT, Nguyen T, Nguyen HH, Kim JH, Hoang LT, Kang C, Phuong LS,

Lim W et al (2005) Avian influenza (H5N1) viruses isolated from humans

in Asia in 2004 exhibit increased virulence in mammals. J Virol 79:

11788 – 11800

Manz B, Schwemmle M, Brunotte L (2013) Adaptation of avian influenza A

virus polymerase in mammals to overcome the host species barrier. J Virol

87: 7200 – 7209

Massin P, van der Werf S, Naffakh N (2001) Residue 627 of PB2 is a

determinant of cold sensitivity in RNA replication of avian influenza

viruses. J Virol 75: 5398 – 5404

Matrosovich M, Tuzikov A, Bovin N, Gambaryan A, Klimov A, Castrucci MR,

Donatelli I, Kawaoka Y (2000) Early alterations of the receptor-binding

properties of H1, H2, and H3 avian influenza virus hemagglutinins after

their introduction into mammals. J Virol 74: 8502 – 8512



837

Matrosovich M, Zhou N, Kawaoka Y, Webster R (1999) The surface

glycoproteins of H5 influenza viruses isolated from humans, chickens, and

wild aquatic birds have distinguishable properties. J Virol 73: 1146 – 1155

Matrosovich MN, Gambaryan AS (2012) Solid-phase assays of

receptor-binding specificity. Methods Mol Biol 865: 71 – 94

Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD (2004) Human

and avian influenza viruses target different cell types in cultures of

human airway epithelium. Proc Natl Acad Sci USA 101: 4620 – 4624

Muchmore EA, Diaz S, Varki A (1998) A structural difference between the cell

surfaces of humans and the great apes. Am J Phys Anthropol 107:

187 – 198

Nelli RK, Kuchipudi SV, White GA, Perez BB, Dunham SP, Chang KC (2010)

Comparative distribution of human and avian type sialic acid influenza

receptors in the pig. BMC Vet Res 6: 4

Nicholls JM, Bourne AJ, Chen H, Guan Y, Peiris JS (2007a) Sialic acid receptor

detection in the human respiratory tract: evidence for widespread

distribution of potential binding sites for human and avian influenza

viruses. Respir Res 8: 73

Nicholls JM, Chan MC, Chan WY, Wong HK, Cheung CY, Kwong DL, Wong MP,

Chui WH, Poon LL, Tsao SW, Guan Y, Peiris JS (2007b) Tropism of avian

influenza A (H5N1) in the upper and lower respiratory tract. Nat Med 13:

147 – 149

Nidom CA, Takano R, Yamada S, Sakai-Tagawa Y, Daulay S, Aswadi D, Suzuki

T, Suzuki Y, Shinya K, Iwatsuki-Horimoto K, Muramoto Y, Kawaoka Y

(2010) Influenza A (H5N1) viruses from pigs, Indonesia. Emerg Infect Dis

16: 1515 – 1523

Nobusawa E, Aoyama T, Kato H, Suzuki Y, Tateno Y, Nakajima K (1991)

Comparison of complete amino acid sequences and receptor-binding

properties among 13 serotypes of hemagglutinins of influenza A viruses.

Virology 182: 475 –485

Ohuchi M, Ohuchi R, Feldmann A, Klenk HD (1997) Regulation of receptor

binding affinity of influenza virus hemagglutinin by its carbohydrate

moiety. J Virol 71: 8377 – 8384

Palese P, Tobita K, Ueda M, Compans RW (1974) Characterization of

temperature sensitive influenza virus mutants defective in neuraminidase.

Virology 61: 397 – 410

Pappas C, Viswanathan K, Chandrasekaran A, Raman R, Katz JM, Sasisekharan

R, Tumpey TM (2010) Receptor specificity and transmission of H2N2

subtype viruses isolated from the pandemic of 1957. PLoS One 5: e11158

Paulson JC, de Vries RP (2013) H5N1 receptor specificity as a factor in

pandemic risk. Virus Res 178: 99 – 113

Paulson JC, Sadler JE, Hill RL (1979) Restoration of specific myxovirus

receptors to asialoerythrocytes by incorporation of sialic acid with pure

sialyltransferases. J Biol Chem 254: 2120 – 2124

Peiris M, Yuen KY, Leung CW, Chan KH, Ip PL, Lai RW, Orr WK, Shortridge KF

(1999) Human infection with influenza H9N2. Lancet 354: 916 – 917

Puzelli S, Di Trani L, Fabiani C, Campitelli L, De Marco MA, Capua I, Aguilera

JF, Zambon M, Donatelli I (2005) Serological analysis of serum samples

from humans exposed to avian H7 influenza viruses in Italy between 1999

and 2003. J Infect Dis 192: 1318 – 1322

Qi X, Qian YH, Bao CJ, Guo XL, Cui LB, Tang FY, Ji H, Huang Y, Cai PQ, Lu B,

Xu K, Shi C, Zhu FC, Zhou MH, Wang H (2013) Probable person to person

transmission of novel avian influenza A (H7N9) virus in Eastern China,

2013: epidemiological investigation. BMJ 347: f4752

Ramos I, Krammer F, Hai R, Aguilera D, Bernal-Rubio D, Steel J, Garcia-Sastre

A, Fernandez-Sesma A (2013) H7N9 influenza viruses interact

preferentially with alpha2,3-linked sialic acids and bind weakly to

alpha2,6-linked sialic acids. J Gen Virol 94: 2417 – 2423

Richard M, Schrauwen EJ, de Graaf M, Bestebroer TM, Spronken MI, van

Boheemen S, de Meulder D, Lexmond P, Linster M, Herfst S, Smith DJ, van

den Brand JM, Burke DF, Kuiken T, Rimmelzwaan GF, Osterhaus AD,

Fouchier RA (2013) Limited airborne transmission of H7N9 influenza A

virus between ferrets. Nature 501: 560 – 563

van Riel D, den Bakker MA, Leijten LM, Chutinimitkul S, Munster VJ, de Wit E,

Rimmelzwaan GF, Fouchier RA, Osterhaus AD, Kuiken T (2010) Seasonal

and pandemic human influenza viruses attach better to human upper

respiratory tract epithelium than avian influenza viruses. Am J Pathol 176:

1614 – 1618

van Riel D, Leijten LM, de Graaf M, Siegers JY, Short KR, Spronken MI,

Schrauwen EJ, Fouchier RA, Osterhaus AD, Kuiken T (2013) Novel

avian-origin influenza A (H7N9) virus attaches to epithelium in

both upper and lower respiratory tract of humans. Am J Pathol 183:

1137 – 1143

van Riel D, Munster VJ, de Wit E, Rimmelzwaan GF, Fouchier RA, Osterhaus

AD, Kuiken T (2006) H5N1 virus attachment to lower respiratory tract.

Science 312: 399

van Riel D, Munster VJ, de Wit E, Rimmelzwaan GF, Fouchier RA, Osterhaus

AD, Kuiken T (2007) Human and avian influenza viruses target different

cells in the lower respiratory tract of humans and other mammals. Am J

Pathol 171: 1215 – 1223

Rimmelzwaan GF, Nieuwkoop NJ, de Mutsert G, Boon AC, Kuiken T, Fouchier

RA, Osterhaus AD (2007) Attachment of infectious influenza A viruses of

various subtypes to live mammalian and avian cells as measured by flow

cytometry. Virus Res 129: 175 – 181

Roberts KL, Shelton H, Scull M, Pickles R, Barclay WS (2011) Lack of

transmission of a human influenza virus with avian receptor specificity

between ferrets is not due to decreased virus shedding but rather a lower

infectivity in vivo. J Gen Virol 92: 1822 – 1831

Robertson MA, Etchison JR, Robertson JS, Summers DF, Stanley P (1978)

Specific changes in the oligosaccharide moieties of VSV grown in different

lectin-resistant CHO cells. Cell 13: 515 – 526

Rogers GN, D’Souza BL (1989) Receptor binding properties of human and

animal H1 influenza virus isolates. Virology 173: 317 – 322

Rogers GN, Paulson JC (1983) Receptor determinants of human and animal

influenza virus isolates: differences in receptor specificity of the H3

hemagglutinin based on species of origin. Virology 127: 361 – 373

Russell CA, Fonville JM, Brown AE, Burke DF, Smith DL, James SL, Herfst S,

van Boheemen S, Linster M, Schrauwen EJ, Katzelnick L, Mosterin A,

Kuiken T, Maher E, Neumann G, Osterhaus AD, Kawaoka Y, Fouchier RA,

Smith DJ (2012) The potential for respiratory droplet-transmissible A/

H5N1 influenza virus to evolve in a mammalian host. Science 336:

1541 – 1547

Russell RJ, Stevens DJ, Haire LF, Gamblin SJ, Skehel JJ (2006) Avian and

human receptor binding by hemagglutinins of influenza A viruses.

Glycoconj J 23: 85 – 92

Russell WMS, Burch RL (1959) The Principles of Humane Experimental

Technique, pp 238 . London: Methuen & Co Ltd

Sauter NK, Bednarski MD, Wurzburg BA, Hanson JE, Whitesides GM, Skehel JJ,

Wiley DC (1989) Hemagglutinins from two influenza virus variants bind to

sialic acid derivatives with millimolar dissociation constants: a 500-MHz

proton nuclear magnetic resonance study. Biochemistry 28: 8388 – 8396

Schauer R, Srinivasan GV, Coddeville B, Zanetta JP, Guerardel Y (2009) Low

incidence of N-glycolylneuraminic acid in birds and reptiles and its

absence in the platypus. Carbohydr Res 344: 1494 – 1500

Schnaar RL, Suzuki A, Stanley P (2009) Glycosphingolipids. In Essentials of

Glycobiology, Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P,



838



Scholtissek, C (1990) Pigs as ‘mixing vessels’ for the creation of new

pandemic influenza A viruses. Med Princ Pract 2: 65 – 71

Scholtissek C, Rohde W, Von Hoyningen V, Rott R (1978) On the origin of the

human influenza virus subtypes H2N2 and H3N2. Virology 87: 13 – 20

Schrauwen EJ, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, Fouchier RA,

Herfst S (2013) Reassortment between Avian H5N1 and human influenza

viruses is mainly restricted to the matrix and neuraminidase gene

segments. PLoS One 8: e59889

Schrauwen EJ, Herfst S, Leijten LM, van Run P, Bestebroer TM, Linster M,

Bodewes R, Kreijtz JH, Rimmelzwaan GF, Osterhaus AD, Fouchier RA,

Kuiken T, van Riel D (2012) The multibasic cleavage site in H5N1 virus is

critical for systemic spread along the olfactory and hematogenous routes

in ferrets. J Virol 86: 3975 – 3984

Schulze IT (1997) Effects of glycosylation on the properties and functions of

influenza virus hemagglutinin. J Infect Dis 176(Suppl 1): S24 – S28

Shelton H, Roberts KL, Molesti E, Temperton N, Barclay WS (2013) Mutations

in haemagglutinin that affect receptor binding and pH stability increase

replication of a PR8 influenza virus with H5 HA in the upper respiratory

tract of ferrets and may contribute to transmissibility. J Gen Virol 94:

1220 – 1229

Shi Y, Zhang W, Wang F, Qi J, Wu Y, Song H, Gao F, Bi Y, Zhang Y, Fan

Z, Qin C, Sun H, Liu J, Haywood J, Liu W, Gong W, Wang D, Shu Y,

Wang Y, Yan J et al (2013) Structures and receptor binding of

hemagglutinins from human-infecting H7N9 influenza viruses. Science

342: 243 – 247

Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y (2006) Avian flu:

influenza virus receptors in the human airway. Nature 440: 435 – 436

Skehel JJ, Stevens DJ, Daniels RS, Douglas AR, Knossow M, Wilson IA, Wiley DC

(1984) A carbohydrate side chain on hemagglutinins of Hong Kong

influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl

Acad Sci USA 81: 1779 – 1783

Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus

entry: the influenza hemagglutinin. Annu Rev Biochem 69: 531 – 569

Sorrell EM, Schrauwen EJ, Linster M, De Graaf M, Herfst S, Fouchier RA (2011)

Predicting ‘airborne’ influenza viruses: (trans-) mission impossible? Curr

Opin Virol 1: 635 – 642

Sorrell EM, Wan H, Araya Y, Song H, Perez DR (2009) Minimal molecular

constraints for respiratory droplet transmission of an avian-human H9N2

influenza A virus. Proc Natl Acad Sci USA 106: 7565 – 7570

Srinivasan K, Raman R, Jayaraman A, Viswanathan K, Sasisekharan R (2013)

Quantitative description of glycan-receptor binding of influenza A virus

H7 hemagglutinin. PLoS One 8: e49597

Sriwilaijaroen N, Kondo S, Yagi H, Takemae N, Saito T, Hiramatsu H, Kato K,

Suzuki Y (2011) N-glycans from porcine trachea and lung: predominant

NeuAcalpha2-6Gal could be a selective pressure for influenza variants in

favor of human-type receptor. PLoS One 6: e16302

Stanley P, Schachter H, Taniguchi N (2009) N-Glycans. In Essentials of



Harbor, NY: Cold spring Harbor Laboratory Press

Steel J, Lowen AC, Mubareka S, Palese P (2009) Transmission of influenza

virus in a mammalian host is increased by PB2 amino acids 627K or 627E/

701N. PLoS Pathog 5: e1000252

Stevens J, Blixt O, Chen LM, Donis RO, Paulson JC, Wilson IA (2008) Recent

avian H5N1 viruses exhibit increased propensity for acquiring human

receptor specificity. J Mol Biol 381: 1382 – 1394

Stevens J, Blixt O, Glaser L, Taubenberger JK, Palese P, Paulson JC, Wilson IA

(2006a) Glycan microarray analysis of the hemagglutinins from modern

and pandemic influenza viruses reveals different receptor specificities. J

Mol Biol 355: 1143 – 1155

Stevens J, Blixt O, Tumpey TM, Taubenberger JK, Paulson JC, Wilson IA

(2006b) Structure and receptor specificity of the hemagglutinin from an

H5N1 influenza virus. Science 312: 404 – 410

Stevens J, Corper AL, Basler CF, Taubenberger JK, Palese P, Wilson IA (2004)

Structure of the uncleaved human H1 hemagglutinin from the extinct

1918 influenza virus. Science 303: 1866 – 1870

Subbarao EK, London W, Murphy BR (1993) A single amino acid in the PB2

gene of influenza A virus is a determinant of host range. J Virol 67:

1761 – 1764

Takemoto DK, Skehel JJ, Wiley DC (1996) A surface plasmon resonance assay

for the binding of influenza virus hemagglutinin to its sialic acid receptor.

Virology 217: 452 – 458

Tharakaraman K, Jayaraman A, Raman R, Viswanathan K, Stebbins NW, Johnson

D, Shriver Z, Sasisekharan V, Sasisekharan R (2013a) Glycan receptor

binding of the influenza A virus H7N9 hemagglutinin. Cell 153: 1486 – 1493

Tharakaraman K, Raman R, Viswanathan K, Stebbins NW, Jayaraman A,

Krishnan A, Sasisekharan V, Sasisekharan R (2013b) Structural

determinants for naturally evolving H5N1 hemagglutinin to switch its

receptor specificity. Cell 153: 1475 – 1485

Thompson CI, Barclay WS, Zambon MC, Pickles RJ (2006) Infection of human

airway epithelium by human and avian strains of influenza a virus. J Virol

80: 8060 – 8068

Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, Recuenco S,

Ellison JA, Davis CT, York IA, Turmelle AS, Moran D, Rogers S, Shi M,

Tao Y, Weil MR, Tang K, Rowe LA, Sammons S, Xu X et al (2012) A

distinct lineage of influenza A virus from bats. Proc Natl Acad Sci USA 109:

4269 – 4274

Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen X, Recuenco

S, Gomez J, Chen L, Johnson A, Tao Y, Dreyfus C, Yu W, McBride R, Carney

PJ, Gilbert AT, Chang J, Guo Z et al (2013) New world bats harbor diverse

influenza A viruses. PLoS Pathog 9: e1003657 . doi: 10.1371/

journal.ppat.1003657

Trebbien R, Larsen LE, Viuff BM (2011) Distribution of sialic acid receptors

and influenza A virus of avian and swine origin in experimentally infected

pigs. Virol J 8: 434

Tumpey TM, Maines TR, Van Hoeven N, Glaser L, Solorzano A, Pappas C, Cox

NJ, Swayne DE, Palese P, Katz JM, Garcia-Sastre A (2007) A two-amino

acid change in the hemagglutinin of the 1918 influenza virus abolishes

transmission. Science 315: 655 – 659

Tweed SA, Skowronski DM, David ST, Larder A, Petric M, Lees W, Li Y, Katz J,

Krajden M, Tellier R, Halpert C, Hirst M, Astell C, Lawrence D, Mak A

(2004) Human illness from avian influenza H7N3, British Columbia. Emerg

Infect Dis 10: 2196 – 2199

Van Hoeven N, Pappas C, Belser JA, Maines TR, Zeng H, Garcia-Sastre A,

Sasisekharan R, Katz JM, Tumpey TM (2009) Human HA and polymerase

subunit PB2 proteins confer transmission of an avian influenza virus

through the air. Proc Natl Acad Sci USA 106: 3366 – 3371

Van Poucke SG, Nicholls JM, Nauwynck HJ, Van Reeth K (2010) Replication of

avian, human and swine influenza viruses in porcine respiratory explants

and association with sialic acid distribution. Virol J 7: 38

Varki A, Sharon N (2009) Historical background and overview. In Essentials of






839

http://dx.doi.org/10.1371/journal.ppat.1003657

Varki NM, Varki A (2007) Diversity in cell surface sialic acid presentations:

implications for biology and disease. Lab Invest 87: 851 – 857

de Vries E, de Vries RP, Wienholts MJ, Floris CE, Jacobs MS, van den Heuvel A,

Rottier PJ, de Haan CA (2012) Influenza A virus entry into cells lacking

sialylated N-glycans. Proc Natl Acad Sci USA 109: 7457 – 7462

de Vries RP, de Vries E, Bosch BJ, de Groot RJ, Rottier PJ, de Haan CA (2010)

The influenza A virus hemagglutinin glycosylation state affects

receptor-binding specificity. Virology 403: 17 – 25

de Vries RP, Zhu X, McBride R, Rigter A, Hanson A, Zhong G, Hatta M, Xu R,

Yu W, Kawaoka Y, de Haan C, Wilson IA, Paulson JC (2014) Hemagglutinin

receptor specificity and structural analyses of respiratory

droplet-transmissible H5N1 viruses. J Virol 88: 768 – 773

Walther T, Karamanska R, Chan RW, Chan MC, Jia N, Air G, Hopton C, Wong

MP, Dell A, Malik Peiris JS, Haslam SM, Nicholls JM (2013) Glycomic

analysis of human respiratory tract tissues and correlation with influenza

virus infection. PLoS Pathog 9: e1003223

Wan H, Perez DR (2007) Amino acid 226 in the hemagglutinin of H9N2

influenza viruses determines cell tropism and replication in human airway

epithelial cells. J Virol 81: 5181 – 5191

Wang H, Feng Z, Shu Y, Yu H, Zhou L, Zu R, Huai Y, Dong J, Bao C, Wen L,

Yang P, Zhao W, Dong L, Zhou M, Liao Q, Yang H, Wang M, Lu X, Shi Z,

Wang W et al (2008) Probable limited person-to-person transmission of

highly pathogenic avian influenza A (H5N1) virus in China. Lancet 371:

1427 – 1434

Wang W, Lu B, Zhou H, Suguitan AL Jr, Cheng X, Subbarao K, Kemble G, Jin H

(2010) Glycosylation at 158N of the hemagglutinin protein and receptor

binding specificity synergistically affect the antigenicity and

immunogenicity of a live attenuated H5N1 A/Vietnam/1203/2004 vaccine

virus in ferrets. J Virol 84: 6570 – 6577

Washington N, Steele RJ, Jackson SJ, Bush D, Mason J, Gill DA, Pitt K, Rawlins

DA (2000) Determination of baseline human nasal pH and the effect of

intranasally administered buffers. Int J Pharm 198: 139 – 146

Watanabe T, Kiso M, Fukuyama S, Nakajima N, Imai M, Yamada S, Murakami

S, Yamayoshi S, Iwatsuki-Horimoto K, Sakoda Y, Takashita E, McBride R,

Noda T, Hatta M, Imai H, Zhao D, Kishida N, Shirakura M, de Vries RP,

Shichinohe S et al (2013) Characterization of H7N9 influenza A viruses

isolated from humans. Nature 501: 551 – 555

Watanabe T, Shinya K, Watanabe S, Imai M, Hatta M, Li C, Wolter BF,

Neumann G, Hanson A, Ozawa M, Yamada S, Imai H, Sakabe S, Takano R,

Iwatsuki-Horimoto K, Kiso M, Ito M, Fukuyama S, Kawakami E, Gorai T

et al (2011) Avian-type receptor-binding ability can increase influenza

virus pathogenicity in macaques. J Virol 85: 13195 – 13203

Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992)

Evolution and ecology of influenza A viruses. Microbiol Rev 56: 152 – 179

Wilson IA, Skehel JJ, Wiley DC (1981) Structure of the haemagglutinin

membrane glycoprotein of influenza virus at 3 A resolution. Nature 289:

366 – 373

de Wit E, Munster VJ, van Riel D, Beyer WE, Rimmelzwaan GF, Kuiken T,

Osterhaus AD, Fouchier RA (2010) Molecular determinants of adaptation

of highly pathogenic avian influenza H7N7 viruses to efficient replication

in the human host. J Virol 84: 1597 – 1606

Xiong X, Coombs PJ, Martin SR, Liu J, Xiao H, McCauley JW, Locher K, Walker

PA, Collins PJ, Kawaoka Y, Skehel JJ, Gamblin SJ (2013a) Receptor binding

by a ferret-transmissible H5 avian influenza virus. Nature 497: 392 – 396

Xiong X, Martin SR, Haire LF, Wharton SA, Daniels RS, Bennett MS,

McCauley JW, Collins PJ, Walker PA, Skehel JJ, Gamblin SJ (2013b)

Receptor binding by an H7N9 influenza virus from humans. Nature 499:

496 – 499

Xu D, Newhouse EI, Amaro RE, Pao HC, Cheng LS, Markwick PR, McCammon

JA, Li WW, Arzberger PW (2009) Distinct glycan topology for avian and

human sialopentasaccharide receptor analogues upon binding different

hemagglutinins: a molecular dynamics perspective. J Mol Biol 387:

465 –491

Xu L, Bao L, Deng W, Dong L, Zhu H, Chen T, Lv Q, Li F, Yuan J, Xiang Z, Gao

K, Xu Y, Huang L, Li Y, Liu J, Yao Y, Yu P, Li X, Huang W, Zhao X et al

(2013) The Novel Avian-Origin Human A (H7N9) Influenza Virus Could be

Transmitted between Ferrets via Respiratory Droplets. J Infect Dis 209:

551 – 556

Xu Q, Wang W, Cheng X, Zengel J, Jin H (2010a) Influenza H1N1 A/Solomon

Island/3/06 virus receptor binding specificity correlates with virus

pathogenicity, antigenicity, and immunogenicity in ferrets. J Virol 84:

4936 – 4945

Xu R, McBride R, Paulson JC, Basler CF, Wilson IA (2010b) Structure, receptor

binding, and antigenicity of influenza virus hemagglutinins from the 1957

H2N2 pandemic. J Virol 84: 1715 – 1721

Yamada S, Hatta M, Staker BL, Watanabe S, Imai M, Shinya K, Sakai-Tagawa

Y, Ito M, Ozawa M, Watanabe T, Sakabe S, Li C, Kim JH, Myler PJ, Phan I,

Raymond A, Smith E, Stacy R, Nidom CA, Lank SM et al (2010) Biological

and structural characterization of a host-adapting amino acid in

influenza virus. PLoS Pathog 6: e1001034

Yamada S, Suzuki Y, Suzuki T, Le MQ, Nidom CA, Sakai-Tagawa Y,

Muramoto Y, Ito M, Kiso M, Horimoto T, Shinya K, Sawada T, Usui T,

Murata T, Lin Y, Hay A, Haire LF, Stevens DJ, Russell RJ, Gamblin SJ

et al (2006) Haemagglutinin mutations responsible for the binding of

H5N1 influenza A viruses to human-type receptors. Nature 444:

378 – 382

Yang H, Carney PJ, Chang JC, Villanueva JM, Stevens J (2013) Structural

analysis of the hemagglutinin from the recent 2013 H7N9 influenza virus.

J Virol 87: 12433 – 12446

Yu Y, Mishra S, Song X, Lasanajak Y, Bradley KC, Tappert MM, Air GM,

Steinhauer DA, Halder S, Cotmore S, Tattersall P, Agbandje-McKenna M,

Cummings RD, Smith DF (2012) Functional glycomic analysis of human

milk glycans reveals the presence of virus receptors and embryonic stem

cell biomarkers. J Biol Chem 287: 44784 – 44799

Zaraket H, Bridges OA, Duan S, Baranovich T, Yoon SW, Reed ML, Salomon R,

Webby RJ, Webster RG, Russell CJ (2013) Increased acid stability of the

hemagglutinin protein enhances H5N1 influenza virus growth in the

upper respiratory tract but is insufficient for transmission in ferrets. J Virol

87: 9911 – 9922

Zeng H, Goldsmith CS, Maines TR, Belser JA, Gustin KM, Pekosz A, Zaki SR,

Katz JM, Tumpey TM (2013) Tropism and infectivity of influenza virus,

including highly pathogenic avian H5N1 virus, in ferret tracheal

differentiated primary epithelial cell cultures. J Virol 87: 2597 – 2607

Zhang Q, Shi J, Deng G, Guo J, Zeng X, He X, Kong H, Gu C, Li X, Liu J, Wang

G, Chen Y, Liu L, Liang L, Li Y, Fan J, Wang J, Li W, Guan L, Li Q et al

(2013a) H7N9 influenza viruses are transmissible in ferrets by respiratory

droplet. Science 341: 410 – 414

Zhang W, Shi Y, Lu X, Shu Y, Qi J, Gao GF (2013b) An airborne transmissible

avian influenza H5 hemagglutinin seen at the atomic level. Science 340:

1463 – 1467

Zhang W, Shi Y, Qi J, Gao F, Li Q, Fan Z, Yan J, Gao GF (2013c) Molecular

basis of the receptor binding specificity switch of the hemagglutinins

from both the 1918 and 2009 pandemic influenza A viruses by a D225G

substitution. J Virol 87: 5949 – 5958

Zhang Y, Zhang Q, Kong H, Jiang Y, Gao Y, Deng G, Shi J, Tian G, Liu L, Liu J, Guan

Y, Bu Z, Chen H (2013d) H5N1 hybrid viruses bearing 2009/H1N1 virus



840

genes transmit in guinea pigs by respiratory droplet. Science 340:

1459 – 1463

Zhang ZF, Fan XH, Li KS, Huang K, Luo DZ, Feng ZB, Wei MY, Guan Y, Chen

HL, Zhang JX (2008) Prevalence of avian influenza virus receptor in

human respiratory tract. Prog Biochem Biophys 35: 1387 – 1393

Zhou J, Wang D, Gao R, Zhao B, Song J, Qi X, Zhang Y, Shi Y, Yang L, Zhu W,

Bai T, Qin K, Lan Y, Zou S, Guo J, Dong J, Dong L, Wei H, Li X, Lu J et al

(2013) Biological features of novel avian influenza A (H7N9) virus. Nature

499: 500 – 503

Zhu H, Wang D, Kelvin DJ, Li L, Zheng Z, Yoon SW, Wong SS, Farooqui A,

Wang J, Banner D, Chen R, Zheng R, Zhou J, Zhang Y, Hong W, Dong W,

Cai Q, Roehrl MH, Huang SS, Kelvin AA et al (2013) Infectivity,

transmission, and pathology of human-isolated H7N9 influenza virus in

ferrets and pigs. Science 341: 183 – 186



841

Documents

Chapter 34, Microbial Lectins: Hemagglutinins, Adhesins ...eskolab.ucsd.edu/advGlyco2016/assigned_reading05-31.pdf · Assigned Reading: Chapter 34, Microbial Lectins: Hemagglutinins,