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Respiratory Syncytial Virus Fusion Protein Interaction with its Cellular Receptor, Nucleolin.
by
Zheng Yuan Daniel Zhu
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Laboratory Medicine and Pathobiology University of Toronto
© Copyright by Zheng Yuan Daniel Zhu 2018
II
Respiratory Syncytial Virus Fusion Protein Interaction with its Cellular
Receptor, Nucleolin.
Zheng Yuan Daniel Zhu
Master of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
2018
Abstract
Respiratory Syncytial virus (RSV) fusion protein (F) interacts with nucleolin during viral fusion.
RNA binding domains 1 and 2 (RBDs) of nucleolin, when expressed as a complex, inhibits RSV
infection in vitro. We examined the interaction between pre- vs. post-fusion F, and whole virus,
with the RBDs complexed and individually. Both RSV Fs co-precipitated RBD1,2. Immobilized
RBD1,2 precipitated both RSV Fs and whole viral particles. Immobilized RBD1 precipitated
post-fusion but not pre-fusion RSV F. RBD1,2 inhibited infection at 20nM when co-incubated
with the virus and the cells for 24 hours. RBD1 inhibited infection to a lesser extent with the
same conditions. RBDs were not able to inhibit infection with coincubation with virus prior to
infection. RBD1,2 interacts with RSV F, showing different preferential binding for one
conformation over another depending on assay conditions, while inhibiting infection at 20nM
with 24 hours of coincubation.
III
Acknowledgments
Supervisor:
Dr. Richard Hegele
Committee Members:
Dr. Jeffrey Lee, Dr. Jeremy Mogridge, Dr. Peter Mastrangelo
Undergraduate lab members:
Alena Tran, Zarian Shahzad
IV
Table of Contents
Acknowledgments III
Table of Contents IV
List of Abbreviations VI
List of Figures VII
List of Appendices VIII
Chapter One: Introduction 1
1.1 Introduction 2
1.2 Pneumoviridae 2
1.3 Respiratory Syncytial Virus 3
1.4 Fusion Proteins 6
1.5 Clinical Impact 10
1.6 Nucleolin 12
1.7 RSV F and Nucleolin interaction model 14
1.8 Hypothesis 15
1.9 Aims 16
Chapter Two: Methods 17
2.0 General Protocols 18
2.1 Expression and Purification of Proteins 20
RBD1, RBD2, and RBD1,2 20
Post-Fusion RSV F 22
Pre-Fusion RSV F 23
Pre-Fusion Specific Antibody 23
SEC pre-fusion and RBD1,2 co-elution 24
2.2 Co-precipitation of RBD1,2 and RSV F 24
RBD1,2 Co-precipitation with RSV F Precipitation 24
RSV F Pull Down with RBD1,2 25
Viral Particle Co-precipitation with RBD1,2 Pull Down 26
2.3 Biolayer interferometry 26
2.4 Cell Culture effects of RBD on RSV infection 27
RBD protein effects on cell metabolic activity 27
Fluorescence quantification of RSV A2 infection 27
Infection inhibition with RBD 28
2.5 RSV F triggering assay 29
Chapter Three: Results 30
3.1 Expression and Purification of Proteins 31
V
RBD1, RBD2, and RBD1,2 31
Pre- and Post-Fusion RSV F 35
Pre-Fusion Specific Antibody 38
Size exclusion chromatography 38
SEC pre-fusion RSV F and RBD1,2 co-elution 39
3.2 Co-precipitation of RBD1,2 and RSV F 41
RBD1,2 Co-precipitation with RSV F Precipitation 41
RSV F Pull Down with RBD1,2 42
Viral Particle Co-precipitation with RBD1,2 Pull Down 45
3.3 Biolayer interferometry 46
3.4 Cell Culture effects of RBD on RSV infection 48
RBD protein effects on cell metabolic activity 48
Fluorescence quantification of RSV A2 infection 49
Infection inhibition with RBD 50
3.5 RSV F triggering assay 55
3.6 NRE Binding to RBD1,2 56
Chapter Four: Discussion 58
4.1 Conclusions 59
4.2 Summary 59
4.3 Cell Surface nucleolin 60
4.4 RSV Pathogenesis 61
4.5 Future Directions 64
Cell Surface Nucleolin and Recombinant RSV F 64
Isothermal Calorimetry 64
Site Directed Mutagenesis 65
Cell Culture Experiments 65
5. References 67
6. Appendix 74
VI
List of Abbreviations
BLI - Biolayer interferometry
CHR - C-terminal heptad repeats
DMEM - Dulbecco's modified Eagle medium
EMEM - Eagle's minimal essential medium
EMSA - Electrophoretic mobility shift assay
FBS - Fetal bovine serum
GP - Glycoprotein
GP1 - Receptor binding subunit
GP2 - Fusion subunit
GAR - Glycine/arginine rich domain
HA - Hemagglutinin
HIV - Human immunodeficiency virus
HIV GP - HIV glycoprotein
NHR - N-terminal heptad repeats
NHS - N-hydroxysuccinimide
PAGE - Polyacrylamide gel electrophoresis
PBS - Phosphate-buffered saline
PS - Penicillin/streptomycin
RBD - RNA binding domain
RSV - Respiratory syncytial virus
RSV F - RSV Fusion protein
RSV G - RSV envelope glycoprotein
RSV SH - RSV short hydrophobic protein
SEC - Size exclusion chromatography
NRE - Nucleolin recognition element
TBS - Tris-buffered saline
-T - Tween 20 (detergent)
VII
List of Figures
1. RSV F Structures 5
2. Class I Fusion Proteins 7
3. Nucleolin domains and RBD1,2 Structure 14
4. Pre-fusion and post-fusion constructs 23
5. RBD expression and purification 31
6. RBD Size exclusion chromatography 32
7. RSV F expression and purification 36
8. RSV F trimerization and size exclusion chromatography 37
9. Co-elution of RDB1,2 and Pre-fusion RSV F on size exclusion chromatography 40
10. Immobilized RSV F co-precipitates RBD 41
11. Input material for RBD immobilization and RSV F precipitation 43
12. Immobilized RBD precipitates RSV F 44
13. Immobilized RBD1,2 co-precipitates RSV A2 virus particles 46
14. Biophysical interaction using biolayer interferometry. 46
15. RBD toxicity assay 49
16. RSV fluorescence quantification 50
17. Cell Culture RSV infection inhibition assay 24hr 51
18. Cell Culture RSV infection inhibition assay microscopy 24hr 52
19. Cell Culture RSV fusion inhibition assay 54
20. RSV virus pre-fusion antibody binding 56
21. Proposed RSV Fusion model 63
VIII
List of Appendices
1. Amino acid sequences of RSV F from RSV A2, pre-fusion construct, and post-fusion
construct
2. Amino acid sequences of RBD1,2, RBD1, and RBD2
3. Antibodies
4. RBD1, RBD2, RBD1,2 plasmid maps
5. RSV F expression plasmids
6. BLItz conditions
7. RBD viability 24 hr incubation
8. Fluorescence assay
9. 24 hr incubation inhibition
10. Cell confluency for 24 hr incubation
11. 2 hr incubation inhibition
12. Cell confluency for 2 hr incubation
13. RBD2 Readthrough Product
1
Chapter One: Introduction
2
1.1 Introduction
Human respiratory syncytial virus (RSV), recently renamed to human orthopneumovirus1, is
enveloped and requires fusion with its host cell membrane in order to infect. Dr. Hegele and his
group had originally identified nucleolin as the fusion receptor in 2011 using a virus overlay
protein binding assay2 and clarified RSV fusion protein (F) was the viral protein involved in the
interaction. They had shown reduction in nucleolin availability through either siRNA or by
antibody binding reduced infectivity of RSV. Furthermore by inducing the expression of
nucleolin in Sf9 cells (which do not natively express nucleolin), they become permissive to RSV
infection2.
My project further characterizes the interaction between nucleolin and RSV F by analysing the
interacting domains using stable recombinant proteins. A better understanding of the protein
docking can shed light into the fusion event between the viral particle and the host membrane,
as well as identify novel targets for intelligent drug design. Utilising cell infection models,
different treatments will be applied to outline possible mechanisms of the interaction between
RSV F and the receptor.
1.2 Pneumoviridae
RSV is in the pneumoviridae family, newly reclassified in the 10th International Committee of
Taxonomy of Viruses report of 20163. Members of this family include orthopneumovirus and
metapneumovirus. The family is characterised by spherical or filamentous enveloped virions
with a helical nucleoprotein core surrounding negative polarity unsegmented RNA genomes.
Their replication occurs entirely in the cytoplasm of the host cells where transcription is
performed by virus encoded RNA polymerase. Transcripted products are either whole genome
copies or individual capped and polyadenylated mRNA. Their host includes avian and
mammalian targets3.
3
1.3 Respiratory Syncytial Virus
RSV has a 15kb genome consists of ten genes coding for eleven proteins, including three
glycoproteins in its envelope: glycoprotein (G), short hydrophobic protein (SH), and the fusion
(F) protein. Of the three glycoproteins only the F protein is mandatory for infection4. RSV G has
been shown to assist in the infection process by binding to various surface proteins5 on the
host membrane, where the viral particle can then access the fusion receptor nucleolin. RSV SH
has been shown to act as a pentameric viroporin which acts as a small molecules channel after
membrane fusion6.
After fusion viral RNA polymerases begin transcribing the negative sense RNA immediately.
During replication, the negative sense RNA is directly transcribed by the viral RNA dependent
RNA polymerase. RSV RNA polymerase has the ability to both transcribe full length
antigenome as well as individual polyadenylated, guanosine capped mRNA segments7. The
mRNA leaves the replication complex to be translated by the host’s cytoplasmic machinery.
Translation of the of the mRNA leads to production of viral proteins in sequential order,
affecting host responses8.
The RSV G protein is a highly variable attachment protein, with no homology with any other
pneumoviridae or paramyxoviridae family member glycoproteins5. RSV G ranges from 289-322
amino acids in length depending on the strain5,9. RSV G has a conserved Heparin-Binding
domain10. Heparin sulfate is commonly found on a variety of cell surfaces and is one of the
attachment receptors for RSV G. Other attachment candidates identified include CX3CR1,
Surfactant Protein A, Annexin II, and lectins DC-SIGN and L-SIGN5, each having a unique
effect upon binding. Despite binding a variety of attachment receptors and stabilizing the virus
on the surface of cells, RSV G is not required for infection4 and has not been shown to play a
role in the fusion process.
4
The RSV F protein is translated as a single peptide F0 which is cleaved at two furin-like
cleavage sites (109 and 137), into covalently bonded smaller F2 and larger F1 while losing a
small interstitial peptide11 (Figure 1A and 1B, Appendix 1). RSV F is a Class I trimeric fusion
protein primed by the cleavage of F012. Currently how RSV F interacts with nucleolin to facilitate
fusion is unclear. RSV F exists in a pre-fusion state where the F1 N-terminal (where the fusion
peptide is located) is compressed and embedded on the inside of the trimer (Figure 1A). The
fusion peptide is followed by N-terminal heptad repeats (NHR). The F1 C-terminal is attached to
the viral membrane by a transmembrane domain preceded by the C-terminal heptad repeats
(CHR). Upon triggering, the F1 N-terminal extends out from its pre-fusion state and is
embedded into the host lipid membrane. After this transient form, driven by the hydrophobicity
of the NHR and the CHR, the two F1 terminals fold over together, where their helical grooves
intertwine, forming the classical 6 helical-bundle and switching to the elongated post-fusion
state5,13(Figure 1B). RSV F has multiple glycosylation sites specific to the strain of origin12,14.
Zimmer et al. have shown that each glycosylation site has its own impact on the syncytium
formation during infection, however they have no effect on the cleavage of F012. It is possible
the glycosylation plays a role in receptor interaction and/or pre-fusion conformation
stabilization. It is believed that the majority of RSV F on the viral envelope exists in the post-
fusion form, and the process by which the pre-fusion conformation induces fusion has yet to be
elucidated. To date, it has been shown that some pre-fusion specific antibodies are capable of
inhibiting viral infection13,15.
Four distinct antigenic domains in the literature have been highlighted on RSV F: sites 0, 1, 2,
and 4. Sites 1, 2, and 4 are available for antibody binding in both pre- and post- fusion RSV F,
whereas site 0 is only accessible in the pre-fusion state, and is located on the axial end, away
from the viral envelope16. Antibody binding to a combination of sites has been shown to prevent
5
pre-fusion triggering12,13,15. Using the same nomenclature, we hope to identify the binding sites
of nucleolin and establish the role of nucleolin in the fusion event.
Figure 1. RSV F X-Ray crystallography structures of Pre- (A) and post-fusion (B) deposited into Protein Data Bank by McLellan et.al.12,16,17
6
1.4 Fusion Proteins
There are four well characterised triggers for Class I trimeric viral fusion proteins: low pH,
receptor binding, proteolytic mediated, and a combination of low pH and receptor binding.
Class I Trimeric Fusion proteins are well studied in Influenza virus a member of the
Orthomyxoviridae family18, which utilizes low pH, Human immunodeficiency virus (HIV) , a
member of the Retroviridae family19, which utilizes receptor binding, Ebola virus which requires
proteolysis to trigger, and avian retroviruses which employs a combination of receptor bind and
low pH20. All Class I fusion proteins undergo triggering of the pre-fusion fusion protein and
structural reconfiguration to achieve fusion (Figure 2).
7
Figure 2. Class I fusion proteins A) Pre-fusion fusion protein is activated by its triggering factors on the viral membrane. B,C) Fusion protein triggers and fusion peptide is injected into host membrane in an intermediate stage. D) Intermediate stage collapses and hemifusion of membranes occurs. E) Fusion protein switches to post-fusion confirmation and membranes are fused.
In the case of low pH triggered fusion proteins, Hemagglutinin (HA) 1 of influenza virus must
first bind sialic acid initiating endocytosis. After entry to endosomes the subsequent
acidification causes reconfiguration of the globular HA1, which originally clamps the fusion
peptide located in the N-terminal of HA2. The reconfiguration exposes the fusion peptide and
8
initiates fusion process, at which point the NHR and CHR drives the conformational change
and remainder of the fusion process. Protonation of various residues is implicated as the driver
for the reconfiguration event.
Receptor binding triggering of fusion proteins have a variety of different mechanisms. In HIV
the glycoprotein (GP) 160 is cleaved to GP120 and GP41 and together facilitates fusion.
GP120 binds CD4 receptor on the host cell and must be associated with a combination of co-
receptors, which induces the ejection of the fusion peptide located on GP41. The NHR then
fold over to associate with the CHR and fusion takes place. This is one example of receptor
mediated fusion process.
Paramyxoviruses and their fusion processes have been described as receptor binding to an
independent viral protein subunit21,22. They require a secondary protein to facilitate the fusion
process. In the case of Parainfluenza virus, the fusion protein is normally not associated with
the attachment protein HN. When HN binds the receptor, exposing activating residues on its
stalk, it then binds the fusion protein and induces the fusion process. This is known as the
provocateur model. On the other hand, in measles and henipavirus the fusion protein is
naturally bound to a pre-fusion stabilizing protein, H and G respectively. The binding of a
receptor to the H or G protein, exposing residues on its stalk, releases the pre-fusion
conformation and allows the fusion process to occur22. This is known as the clamp model. Both
these models require extensive interaction with a secondary attachment protein to facilitate the
fusion process when the virus is situated on the host membrane. However, unlike the
paramyxoviridae family, pneumoviridae do not require the attachment protein to facilitate
fusion4,21,23.
Other receptor mediated triggering can occur through the interaction of a single viral fusion
protein with a single host receptor protein, such as in case of many retroviruses and
9
coronaviruses. On the other hand, receptor mediated triggering can involve multiple viral
proteins in the case of herpesviruses20.
Avian retroviruses fusion proteins have been shown to exhibit binding with fusion receptors
through the receptor binding subunits, followed by conformation change in low pH, inducing the
exposure of the fusion subunit and the fusion event20. The requirement for the receptor is the
key difference to the low pH triggered model. In the case of Influenza, it has been shown to be
able to fuse in low pH liposomes in the absence of receptors.
Ebola virus has a proteolytic triggering system. The GP is divided into two subunits: the
receptor binding subunit (GP1), and the fusion subunit (GP2). Ebola GP on the viral membrane
must undergo proteolysis to remove the clamped state of the fusion subunit. GP1 must be
digested by a variety of proteases, cathepsin B and cathepsin L, in a process know as priming.
The clamp GP1 must go from being a 130 kDa subunit to 19 kDa before GP2 is released and
the fusion can be triggered. Although all Class I and Class II fusion proteins require proteolysis
of the fusion protein to become active, this extensive proteolytic priming is exclusive to this type
of fusion protein.
Besides Class I fusion proteins, there are Class II and Class III viral fusion proteins. Like Class
I, Class II requires proteolytic processing of the fusion protein to become active. Class II fusion
protein is composed, in majority, out of beta sheets and is oriented parallel to the viral
membrane and exists as dimers. In Class II fusion proteins the fusion peptide is located in the
central regions and low pH has been described as its fusion peptide activator. Class III fusion
proteins do not require proteolytic processing and have a variety of dominant structures and
orientations, employing a combination of Class I and Class II fusion protein structures and
domains20.
10
1.5 Clinical Impact
RSV is a global pathogen affecting individuals of all ages, presenting with an increased risk of
severe lower respiratory tract infections in infants and the elderly24. Currently treatment is
supportive care and vaccines are in development. Ribavirin is the only approved therapeutic,
however because of its severe side-effects and the possible hazards in its administration, it is
infrequently used. Other treatment options such as bronchodilators and corticosteroids have
varying evidence supporting their efficacy25. Administration of oxygen or oxygen mixtures in
severe cases, may reduce morbidity but there is a lack of evidence suggesting their efficacy in
reducing hospitalization time of patients, a key indicator of treatment success26. The current
leading candidates of RSV therapeutics are a fusion inhibitor, and a nucleoside analogue27.
Cox and Plemper have described multiple fusion inhibitors, however these candidates have
already presented with the emergence of resistant strains; on the other hand, nucleoside and
nucleotide analogues have historically presented with severe adverse effects and clinical use in
pediatric and neonatal populations is difficult. Research into other inhibitors of the viral RNA
polymerase complex is still in early stages of development28.
Palivizumab is a human monoclonal antibody against RSV F used as prophylaxis for
populations at risk for severe disease29. Palivizumab is a neutralizing antibody targeting epitope
site 4 on both pre- and post-fusion RSV F and it has been shown to reduce the occurrence of
severe infections in treated patients29. There is however evidence that resistance conferring
mutations in the Palivizumab epitope on RSV F are selected for30.
In the 1960’s formalin inactivated RSV was tested as a vaccine candidate in humans. The
clinical trials resulted in an exaggerated disease response when vaccinated individuals were
exposed to the virus. This led to elevated immune infiltration of the lungs, severe pathology,
and even death31. Research suggests non-neutralizing antibodies that do not inhibit viral entry
11
and replication can elicit an exaggerated immune response leading to extensive lung damage
in the patients who were vaccinated32.
Currently the leading strategy for vaccine development involves using different forms of
recombinant RSV F coupled with an adjuvant33. However, recently multiple vaccines utilising
recombinant forms of post-fusion RSV F have failed to reduce viral load and prove effective in
clinical trials34. Despite setbacks, researchers continue to press forward with vaccine
development as the primary goal in the RSV field35-39. Although RSV F dominates as the
antigenic candidate35,36, other strategies involving RSV G37, RSV M238 (an RNA transcription
and replication factor40), and live attenuated viruses are being explored39. However the original
concerns with the other candidates remain; RSV G’s hypervariable regions, RSV M2 is an
intracellular protein, and the storage and production challenges of live attenuated vaccines.
Furthermore, Leemans et al. have shown that RSV F binding to IgG antibodies, regardless of
epitope, induces protein internalization in a variety of cell models41. The consequences of this
effect can greatly impact the efficacy of any vaccine development.
The negative impact of non-neutralizing antibodies on the outcome of disease and the lack of
evidence supporting the mounting of a lasting inhibitory IgA immune response to vaccination is
of concern in the development of these novel vaccines34. Furthermore, fusion inhibitors and
screening of therapeutics without elucidation of viral pathways only leads to emergence of
resistant strains. Better understanding of binding characteristics between RSV F and its
receptor, nucleolin, would be crucial in the engineering of more precise vaccines and
therapeutics to combat severe infections.
12
1.6 Nucleolin
Human nucleolin is encoded by the NCL gene and is mainly distributed in the nucleus.
Nucleolin has three N-terminal acidic stretches, four RBDs in the central region, followed by C-
terminal glycine/arginine rich (GAR) domain (Figure 3A). Nucleolin is known to have multiple
functions, including protein shuttling, transcriptional modulation, nucleic acid stabilization,
ribosomal synthesis, and cell signalling42. Nucleolin has been shown to bind DNA upstream of
rRNA transcription initiation sequences, bind histone H1, as well as be involved in chromatin
remodelling43. These functions have been suggested to be associated with its ribosomal
synthesis and transcription modulation capabilities. Nucleolin has been shown to bind pre-
rRNA in stem loop regions and correlate with chaperoning proper assembly of pre-ribosomal
subunits and their nuclear-cytoplasmic transport, and is required for the first step in pre-rRNA
processing. Nucleolin has DNA dependent ATPase and autodegradative capabilities, as well as
helicase activity, interaction with replication protein A and telomerase, all associated with
nucleolin’s putative roles in cell growth and replication. Nucleolin has been shown to interact
with telomerase, Rad51, and p53, as well as functioning as a downstream effector for Hsp70,
involving nucleolin in DNA damage repair, cell stress response, and cell senescence. Nucleolin
has cell cycle dependent effects, and depletion has been shown to arrest cell cycle in the G2
phase under specific conditions preventing mitosis44.
Nucleolin has a bipartite nuclear localization signal, and the mechanism for its relocation to the
cytosol and cell surface is believed to involve a combination of its complex post translational
modifications43. Studies have shown nucleolin is present in the cytosol mostly in vesicles and
transported to the cell surface independently of the reticulum-Golgi complex45. The presence of
cell surface nucleolin is dependent on an intact actin cytoskeleton and has been shown to be
able to internalize extracellular material, while associated with a variety of proteins45. Nucleolin
has been shown to have different states of phosphorylation, methylation, and ADP-ribosylation,
13
as well as complex N-linked and O-linked glycosylation patterns, outside of the nucleus44,46.
These different post-translational modifications have been shown to affect the function and
localization of nucleolin46.
Cell surface nucleolin has been implicated in the infection of variety of viruses42,43,47. For
example, HIV GP120 V3 loop can bind bind a 95kDa nucleolin on the cell surface of CD4+
cells47,48. The domains which associate with the cell surface, and the orientation of cell surface
nucleolin have yet to be discovered. Cell surface nucleolin has been associated with multiple
proteins44,46, and despite the lack of transmembrane domains, nucleolin has been shown to be
associated with flotillin, a multifunctional lipid raft protein49. Furthermore the domains of
nucleolin which interact with RSV F have not been elucidated.
Nucleolin has four centralized RNA binding domains (RBD 1, 2, 3, 4). The two most N-terminal
of the RBD (1,2) can be expressed recombinantly (Figure 3B, Appendix 2). RBD1,2 is well
characterised and forms a stable protein in solution50. RBD1,2 binds a ([U/G]CCCG[A/G]) RNA
hairpin identified as the nucleolin recognition element (NRE)51, and the physiological binding of
RNA at the cell surface has not been studied. Our lab has previously shown inhibition of RSV
infection when viral particles are co-incubated with RBD1,2 prior to infection52.
14
A
B
Figure 3. Nucleolin domains and RBD1,2 Structure. A) Identified domains of nucleolin B) NMR-Spectroscopy structure of RBD1,2 from Protein Data Bank by Arumugam et.al.36
1.7 RSV F and Nucleolin interaction model
As we begin to better understand the nature of cell surface nucleolin, we can begin to
hypothesize the interactions taking place with RSV F. When RSV F reaches the surface of the
15
cell, extracellular enzymes cleave the interstitial peptide priming the class I fusion protein.
However unlike other class I fusion proteins in the paramyxovirus family, RSV, like other
members of the pneumoviridae family, does not require a secondary viral protein to facilitate
fusion22. The metastable prefusion conformation may only require the binding to its cell surface
receptor to facilitate fusion, similar to metapneumovirus23. However unlike metapneumovirus
receptor alpha(V)-beta(1) integrin, which is classically found on the lipid bilayer, the functional
state and orientation of nucleolin in the lipid rafts is not well studied. If interactions stabilizing
the fusion event are occurring with other proteins present in the lipid rafts, the mechanisms of
viral infection will be complicated. My research hopes to elucidate the role of nucleolin in the
fusion event, either as a guide for RSV F to interact with the lipid rafts or the plasma membrane
itself, or nucleolin as a trigger for RSV F and being the sole interacting partner during the fusion
process (Figure 3). The simplest model for RSV fusion would begin with the attachment of the
viral particle to the host cell. RSV G would bind one or a combination of its receptors to
facilitate the stabilization of the viral particle and trigger downstream effects. Viral particle RSV
F would then come in contact with cell surface nucleolin. Cell surface nucleolin would have
RBD1,2 in its central region oriented towards the extracellular lumen. The interaction of RSV F
with RBD1,2 would cause a conformational change, exposing the fusion peptide and allowing it
to insert into the host membrane, followed by the rearrangement of the CHR and NHR causing
RSV F to take on the post-fusion conformation and fusing the viral and cell membranes. In
order to show this, the interacting domains between RSV F and nucleolin must be better
studied, and the nature of the interaction will have to be elucidated.
1.8 Hypothesis
Pre-fusion RSV F binds the RBD1,2 regions of nucleolin, triggering the elongation of the protein
trimer, causing fusion of the viral envelope with the cell membrane.
16
1.9 Aims
1. Synthesize recombinant pre- and post-fusion forms of RSV F, as well as RBD1, RBD2,
and RBD1,2.
2. Characterize the interaction between RBD1, RBD2, RBD1,2 and the two conformations
of RSV F (pre-fusion and post-fusion)
3. Test RSV fusion inhibition in a cell infection model using recombinant RBD1, RBD2,
and RBD1,2.
17
Chapter Two: Methods
18
2.0 General Protocols
Polyacrylamide gel electrophoresis (PAGE)
All PAGE (SDS and BlueNative) was done using ThermoFisher Scientific electrophoresis
system, buffers, and pre-cast 4-20% agarose gradient gels. All proteins were loaded in their
recommended loading buffers. SDS-PAGE was performed using Laemilli buffer with 10% beta-
mercaptoethanol to dilute protein samples and BlueNative PAGE was performed in Coomassie
G250 sample buffer. When required, boiling the samples in sample buffer was conducted at
90oC for 5 minutes. For better resolution of smaller proteins, running time was 135 minutes at
125V.
Western Blotting
Pre-cast gels were transferred to ThermoFisher iBlot Nitrocellulose membranes using their iBlot
Dry blotting System. Transferred blots were either stored in -20oC until blocking or blocked
immediately. The membranes were blocked at 4oC in 5% w/v skimmed milk powder diluted in
Tris-buffered saline (TBS) with 0.1% Tween20 (-T) overnight, or at room temperature for half
an hour. Blocking solution was washed for five minutes using 4oC TBS-T 0.1%. Primary
antibodies (Appendix 3) were diluted in TBS-T 0.1% with 1% w/v milk and added to the
membranes for one hour at room temperature or at 4oC overnight. Primary antibody solutions
were removed and membranes were washed three times, five minutes per wash, in 4oC TBS-T
0.1%. Secondary antibodies (Appendix 3) were diluted in the same diluent as primary
antibodies and added to the membranes for one hour at room temperature. Secondary
antibody solutions were removed and membranes were washed three times as previously
described. ThermoFisher SuperSignal West Pico Chemiluminescent Substrate was used
according to their protocol and imaged using FusionFx7 imager from MBI laboratory
equipment.
19
Cell Culture
Frozen stock of cells (HEK293T or HEp-2) was thawed in 37oC water bath from liquid nitrogen
to perform the following experiments. Thawed cells were added to Corning 225cm2 flasks
containing 45mL of pre-conditioned (incubated in 37oC with 5% CO2) Dulbecco's Modified
Eagle Medium (DMEM) or Eagle's minimal essential medium (EMEM), with added 5% fetal
bovine serum (FBS) and 1% penicillin and streptomycin (PS), for HEK293T or HEp-2 cells
respectively. Thawed cells were allowed to adhere to the flasks for eight hours or overnight in
37oC incubators with 5% CO2 before the media solution was replaced. Cell Cultures were
allowed to grow to confluency before the media solution was removed and the cells were
washed with 37oC phosphate buffered saline (PBS). 9mL of Trypsin EDTA 0.25% was used at
37oC with 5% CO2 to fully dissociate the cells and 18mL of corresponding media solution was
added to terminate the trypsinization. Cells were counted using a Z series Beckman Coulter
cell counter and diluted in their corresponding media solution to the desired concentrations in
Corning 225cm2 flasks or 96x well plates for further use. Remaining cells were centrifuged at
500 x g for five minutes and the supernatant was removed and replaced with 0.5mL of fresh
media. The pellet was resuspended and 9mL of freezing solution (10% DMSO, 20% FBS in
corresponding media) was added slowly while mixing the cells gently. The cell suspension was
aliquoted to liquid nitrogen tubes and frozen in -80oC in the cell freezing receptacle overnight.
Frozen cells were moved to liquid nitrogen the following day.
Virus Propagation
HEp-2 cells in Corning 225cm2 flasks were prepared at 60% confluency 24 hours prior to
infection. RSV A2 virus stock from American Type Culture Collection was thawed in 37oC water
bath from liquid nitrogen to infect the prepared cells. The thawed virus was diluted in 35mL of
EMEM, with 5% FBS and 1%PS, and added to prepared HEp-2 cells. The flask was gently
agitated at four hours to encourage infection of precipitated viral particles. At 72 hours after
20
infection,10mL of media solution was added to the flasks, while another flask of 60% confluent
HEp-2 cells was prepared for continued viral propagation. At 96 hours the media was
harvested and concentrated to 5mL in Amicon Ultra-15 100kDa centrifugal filtration units.
Concentrated virus was diluted in media solution and added to the prepared HEp-2 cells. This
passaging was repeated a minimum of three times after thawing the virus to achieve a
consistent viral concentration each harvest.
Plasmid preparation
RBD or RSV F plasmids were used to transform NEB 5-alpha Competent E.coli by New
England Biolabs. Transformed bacteria were plated on Biobasic LB-agarose plates with
ampicillin and chloramphenicol or just ampicillin for RBD or RSV F plasmids respectively.
Isolated colonies were grown in LB broth with respective antibiotics at recommended volumes
for the following Thermo HiPure Plasmid Filter Maxiprep or Miniprep protocol. Plasmids
prepared was quantified and analysed using 280/260 nm UV readings on our Tecan Infinite
M200 plate reader and stored at -20oC.
2.1 Expression and Purification of Proteins
RBD1, RBD2, and RBD1,2
Human RBD1,2 as described by Arumugam et al.50 (Appendix 2) was expressed in the Rosetta
2 E.coli strain, using the pET-21a inducible plasmid system (Appendix 4). The E.coli was grown
in Novagen OvernightExpress Autoinduction media and harvested after 24 hours incubation at
37oC with agitation. After lysing the cell pellet using Sigma-Aldrich CelLytic B, Sigma-Aldrich
Protease Inhibitor Cocktail, benzonase, and lysozyme, the protein was purified using the C-
terminal His-Tag on a nickel column, and quantified using a Bradford assay. Purity and size
21
was assessed using SDS-PAGE, as well as Blue-Native PAGE, combined with western blotting
or coomassie blue staining respectively, and size exclusion chromatography (SEC).
Previous work in our lab has shown that RBD1,2 effectively binds NRE using an electrophoretic
mobility shift assay (EMSA), while containing no RNA contaminants from the purification and
will not interfere with the interaction. The protein has been shown to be able to withstand urea
denaturation during purification washes on the nickel column as described by Arumugam et
al.50, however our lab has shown that there is less NRE binding capacity with RBD1,2 that has
been urea washed. Instead I developed a series of sequential imidazole washes, to purify the
RBD, which had overall lower yield but maintained purity and had better binding capacity to
NRE. After lysing the cell pellet using Sigma-Aldrich CelLytic B, Sigma-Aldrich Protease
Inhibitor Cocktail, benzonase, and lysozyme, the lysate was loaded onto a GE 1.5mL
HisGravitrap columns. The lysate was washed using 5mL of PBS, followed by 5mL of 5mM,
10mM, 25mM, 50mM, and 2.5mL of 100mM imidazole in PBS solutions, sequentially. RBDs
were then eluted using 200mM, 300mM, and 500mM imidazole in PBS solutions. 200mM and
300mM eluates were the most pure samples and the eluates were then run on an Amicon
Ultra-15 50kDa Centrifugal Filter unit and through Superdex 200 SEC column to further purify
the protein.
RBD1 and RBD2 was expressed separately using the same system at similar yields, although
RBD2 consistently yielded less than RBD1, and RBD1,2. All RBDs were dialysed in Thermo
Scientific Slide-A-Lyzer 10kDa dialysis tubes and stored in buffered (phosphate 10mM, tris
50mM, or HEPES 25mM) solutions, pH 7.4, with 300mM NaCl and 50mM imidazole. Any
further concentration was conducted using Amicon Ultra-15 Centrifugal Filter units with 10kDa
cut-off for RBD1,2 and 3kDa for RBD1 and RBD2. RBD1 at concentrations above 1.5mg/mL
and when stored in solutions with less than 50mM imidazole would precipitate out of solution.
22
To reduce precipitation, and to keep samples consistent the same diluent was used for all three
RBDs. All proteins were stored at 4oC.
Post-Fusion RSV F
RSV F ectodomain (residues 1-513) gene constructs (Figure 4, Appendix 1), cloned into
expression plasmids (Appendix 5), were ordered through GeneArt Life Technologies and
expressed in HEK293T cells using a CMV promoter. Post-fusion RSV F contains 3 naturally
occurring expression enhancing mutations (P102A, I379V, and M447V) and to prevent
aggregation, have the first 9 residues of the fusion peptide (137 to 146) deleted as described
by McLellan et al12. The cells were transfected using polyethylenimine in 2L Corning
CellSTACK chambers according to protocols described by Aydin et al.53. The protein has a C-
terminal His-Tag and Strep-Tag II preceded by a thrombin cleavage site. The protein
expressed was secreted into media after four days of expression and all cell debris and
aggregates were removed by spinning the media solution at 103x g, followed by concentration
and media solution was replaced by TBS using a Centramate Tangential Flow system. RSV F
was purified from media solution concentrate using both nickel and StrepTactin columns and
successfully cleaved using recombinant thrombin from Novagen in Thrombin Cleavage Buffer
(at 10x 200 mM Tris-HCl, pH 8.4, 1.5 M NaCl, 25 mM CaCl2) at 1 U to 100μg of RSV F for 24
hours at room temperature. Column Eluates were run on Superdex 200 SEC column to further
purify the protein and/or to remove thrombin and cleaved tags. Purified protein was dialysed in
Thermo Scientific Slide-A-Lyzer 10kDa dialysis tubes and stored in buffered (phosphate 10mM,
tris 50mM, or HEPES 25mM) solutions, pH7.4, with 300mM NaCl. Any further concentration
was conducted using Amicon Ultra-15 Centrifugal Filter units with 50kDa cut-off and RSV F
was stored at 4oC. Eluates and flowthrough from each column and concentration step were run
on SDS-PAGE and blotted for RSV F using anti-HisTag, Palivizumab, and anti-StrepTag
antibodies to assess yield and protein lost from each step.
23
Pre-Fusion RSV F
RSV F ectodomain (residues 26-503) with three pre-fusion stabilizing substitutions (Figure 4,
Appendix 1) was expressed using the same system as the post-fusion protein (Appendix 5).
Pre-fusion has two alpha 4-alpha 5 helix hinge stabilizing mutations (S215P and N67I), and a
repulsion charge reduction mutation (E487Q) preventing the extension of alpha 4-alpha 5 helix
hinge and the triggering to the post-fusion conformation as described by Krarup et al54. The
furin cleavage sites and the interstitial peptide was replaced with a (GSGSGR) spacer to
increase expression. Trimerization was enhanced with a C-terminal fibritin domain (Appendix
1). C-terminal His-Tag and Strep-Tag II was also present preceded by a thrombin cleavage
site.
Figure 4. Pre- and post-fusion RSV F constructs compared to the RSV A2 wildtype RSV F. Not including amino acid substitutions P102A, I379V, and M447V on both constructs and S215P, N67I, and E487Q on the prefusion construct.
Pre-Fusion Specific Antibody
D25, anti-pre-fusion complement determining regions12,55 were cloned into heavy and light
chain Fabs and co-expressed in HEK293T cells. D25 binds site 0 on the axial terminal of pre-
fusion RSV F12. D25 Fab incorporated a FLAG-Tag on the heavy chain for further purification
and concentration. AM14, another pre-fusion specific antibody55 was purchased through
24
Creative Biolabs. AM14 laterally targets a combination of sites 0 and 4 in an intermediate
region between the apex and the viral membrane56. Both conformations of RSV F were blotted
onto nitrocellulose membranes, blocked, and detected using either Palivizumab, AM14, or D25
Fab.
SEC pre-fusion and RBD1,2 co-elution
Pre-fusion RSV F at 100 μg/mL was incubated with RBD1,2 at 33 μg/mL for 24 hr at 4oC in
previously described TBS and run on the SEC column.
2.2 Co-precipitation of RBD1,2 and RSV F
RBD1,2 Co-precipitation with RSV F Precipitation
RBD1,2 was co-precipitated while pulling down pre- and post-fusion RSV F using Streptactin
MagStrep XT magnetic beads from IBA. The RBD proteins were co-incubated for 2 hours at
room temperature at a 3:1 molar ratio with either pre- or post-fusion RSV F at 100 μg/mL and
the protein mixtures were added to the equilibrated beads. RBD1,2 also was incubated with
and without 10ng of NRE 30 minutes prior to mixing with RSV F proteins to assess whether
NRE played a role in the interaction between the two proteins. 20µL of the beads-containing
solution were first resuspended in 200µL Strep-tag® washing buffer (100 mM Tris/HCl, pH 8,
150 mM NaCl, 1 mM EDTA) and the buffer was removed using a magnetic rack. Equilibration
was repeated 2 times before the protein samples were added. The beads in the sample were
allowed to bind at room temperature for 30 minutes with occasional mixing, vortex was not
used. The supernatant was removed using the magnetic stand. The beads were washed three
times by resuspending them in 100µL Strep-tag® washing buffer, and removing the
supernatant using the magnetic rack. RBD1,2 had a slight affinity for the sepharose matrix of
the beads. To remove this nonspecific binding, the molarity of NaCl in the manufacturer’s wash
25
buffer was raised to 400mM and 0.01% Tween20 was added, resulting in the elimination of
nonspecific binding of RBD1,2. RSV F was eluted from the beads 25µL Buffer BX and RDB1,2
was co-eluted with both RSV F. Eluates were run on SDS-PAGE and visualised on a Western
blot using Palivizumab and H250 for RSV F and RBD proteins respectively.
RSV F Pull Down with RBD1,2
To extend the previous result, the reciprocal precipitation was performed. Pre- and post-fusion
RSV F was pulled down using RBD1,2 crosslinked Pierce N-hydroxysuccinimide (NHS)
magnetic beads from ThermoFisher Scientific. The beads were left at room temperature for 20
minutes to equilibrate and mixed thoroughly before aliquoting 30µL into a microfuge tube. The
supernatant was removed using a magnetic stand and a micropipette. 1mL of 4oC 1mM
hydrochloric acid solution was added to the tube and mixed by inversion. The supernatant was
removed using the magnetic stand. Immediately after, room temperature RBD1, RBD2,
RBD1,2, and milk proteins in excess with Coupling Buffer (0.15M Triethanolamine, 300mM
NaCl pH 8.3) were crosslinked to NHS beads for one hour at room temperature while mixing
every five minutes during the first 30 minutes of the incubation, and for the remaining time,
mixing the tube every 15 minutes until incubation is complete. The supernatant was removed
using a magnetic stand and the beads were washed at room temperature with 0.4mL of
Blocking Buffer A (0.5M Ethanolamine, 300mM NaCl pH 8.3) and then with Blocking Buffer B
(0.1M NaAc 300mM NaCl pH 4.0) while discarding the supernatant using the magnetic stand
after each wash. The beads were then incubated in 0.4mL of Blocking Buffer A for 15 minutes
at room temperature and the supernatant was discarded using the magnetic stand. The beads
were washed using 0.4mL of Blocking buffer B and the supernatant was discarded using the
magnetic stand. The beads were then washed at room temperature with 0.4mL of Blocking
Buffer A and then with Blocking Buffer B while discarding the supernatant using the magnetic
stand after each wash. The beads were washed using 0.4mL of TBS and the supernatant was
discarded using the magnetic stand. The generated beads were incubated with 10μL of
26
100μg/mL in TBS of pre- and post-fusion RSV F as well as a 1:1 molar ratio mixture of pre- and
post-fusion at 200μg/mL in TBS for 2 hours at room temperature the supernatant was
discarded using the magnetic stand. The beads were washed three times using 200μL of TBS
and the supernatant was discarded using the magnetic stand after each wash. Bound proteins
were eluted by boiling in SDS for ten minutes.
Viral Particle Co-precipitation with RBD1,2 Pull Down
Viral particles were co-precipitated while pulling down RBD1,2 using nickel magnetic beads
from GE. The beads were mixed by inversion and 25µL was aliquoted into microfuge tubes.
The supernatant was removed and 100µL of Wash Buffer (20mM sodium phosphate, 300mM
NaCl, pH 7.4) was used to wash the beads by inversion. RBD1,2 was co-incubated with RSV
A2 virus particles for two hours at room temperature at a 2 pM : 1 focus forming unit (FFU)/mL
ratio. The RBD1,2 and virus were added to the beads and allowed to bind for 30 min with
mixing at room temperature. The supernatant was removed using a magnetic rack and the
beads were washed using 200µL of Wash Buffer. The supernatant was removed and the wash
was repeated two more times. The bound proteins were removed using 25µL of Elution Buffer
(20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4) for ten minutes at room
temperature. Initially viral particles bound nonspecifically to the magnetic beads, but by
including 20mM imidazole in the Wash Buffer, the nonspecific binding was eliminated.
2.3 Biolayer interferometry
The affinity of RSV F and the RBD1,2 was assessed using biolayer interferometry (BLI) on the
Fortebio BLItz system. RBD1,2 was loaded onto the Anti-Penta-His biosensors at 25 and 50
μg/mL and the RSV F proteins was added at varying concentrations to determine the Kd
(Appendix 6). Furthermore, RSV F was loaded onto the StreptAvidin biosensors at 50 and 100
μg/mL and RBD1,2 was added at varying concentrations (Appendix 6). Baseline setting times,
27
loading times, secondary baseline settings times, association time, and disassociation times
were adjusted in attempt to maximize the binding signal. It was anticipated that pre-fusion RSV
F will have faster binding rates with the RBD1,2 proteins and/or slower dissociation rates in
concordance with the competitive pull down assay depending on the buffers used.
2.4 Cell Culture effects of RBD on RSV infection
RBD protein effects on cell metabolic activity
RBD proteins were incubated with HEp-2 cells at varying concentrations for 24 hours and
metabolic activity of the cells were assessed using a Promega MTT. HEp-2 cells were seeded
at 5000 cells/well on Corning 96 well Microplates in media solution previously described and
allowed to adhere and grow for 24 hours prior to the experiment. RBD proteins were diluted to
100µL in EMEM with 5%FBS and 1%PS. The RBD proteins were added to the cells and
incubated at 37oC with 5%CO2 for 24 hours. The MTT assay was performed by adding 15µL of
the Dye Solution to each well. The Formazan product was allowed to develop for one hour at
37oC with 5%CO2, and then the 100µL Solubilization Solution/Stop Mix was added to each well.
Formazan concentration was measured as absorption at 570 nm on a Tecan Infinite
M200ProTM plate reader (Appendix 7).
Fluorescence quantification of RSV A2 infection
RSV A2 was produced by infecting HEp-2 cells with viral stock and left to propagate for 96
hours in EMEM with 5%FBS and 1%PS as previously described. The media was harvested,
spun at 1500 x g for 20 minutes to remove cell debris and the supernatant was used as fresh
virus or concentrated using Amicon Ultra-15 Centrifugal Filter Units with 100kDa cut-off to 5mL.
Both the fresh and concentrated virus was titrated and FFU/mL was calculated by manually
counting FFU in the highest dilution wells still containing infection. HEp-2 cells were seeded at
28
5000 cells/well and grown for 24 hours prior to the infection at 37oC and 5% CO2 in media
solution previously described, on a Corning 96 well black clear-bottom microplate. Virus was
diluted in EMEM with 2.5%FBS and 1%PS and was added to the HEp-2 cells for one hour.
Unbound virus was removed using a warm PBS wash. Infection was propagated for 24 hours
after the wash, in EMEM with 5%FBS and 1%PS, before fixing with a 1:1 solution of -20oC
methanol acetone for 15 minutes at room temperature. Plates were blocked using 5% w/v milk
in TBS-T 0.1%. Infection was visualized using a 1:200 dilution of NCL-RSV3 primary antibody
cocktail from Leica biosystems, an anti-RSV triple mouse monoclonal cocktail which binds RSV
F, RSV N, and RSV phosphoprotein, coupled with a 1:2000 dilution of Alexa Fluor 488, an anti-
mouse fluorescence secondary antibody (A-11001 by ThermoFisher). Primary and secondary
antibodies were incubated for 1 hour each with agitation before being washed three times in
4oC TBS-T 0.1% for 5 min each. The virus titration was also used to measure mean
fluorescence with a Tecan Infinite M200ProTM plate reader (ex. 488 nm, em. 519 nm) and the
two values were compared to establish a viral concentration fluorescence curve (Appendix 8).
Infection inhibition with RBD
The inhibition of infection by RBD1,2 has previously been demonstrated by our lab52. RBD1,2
had been shown to inhibit RSV infection in the picomolar concentration range, when incubated
with the virus at 37oC for 3 hours prior to challenging HEp-2 cells. The virus quantification by
mean fluorescence has been previously described57. The virus and RBD1,2 mixture was added
to the cells for 1 hour at 37oC in EMEM with 2% FBS and 1% PS, at which point unbound virus
was washed off using warm PBS. RBD1,2 at the tested concentrations with EMEM at 2% FBS
and 1% PS had then been replaced and the cell culture was incubated for an additional 24
hours and fixed using cold 75% acetone in PBS for 5 min. Cells were incubated with a 1:10
dilution of RSV3 in PBS with 0.1% Tween20 and 10% FBS for 30 minutes at room temperature.
Cells were washed again as before and incubated with 1:400 dilution (12.5 μg/mL) of Alexa
29
Fluor 488 anti-mouse and a 1:200 dilution of ToPro3 in PBS with 0.1% Tween 20 and 10%
FBS. Then, after a final washing, fluorescence was detected (Appendix 9).
The experiment was repeated using RBD1, and RBD2, as well as Palivizumab as the viral
inhibitor control cell confluency was detected using brightfield microscopy (Appendix 10).
To assess the state of the cell surface bound but unfused virus, RBD1, RBD2, and RBD1,2 at
the tested concentrations were incubated with live RSV A2 strain virus for 60, 90, 120, and 150
minutes at room temperature and 37oC and then added to live HEp-2 cells, prepared 24 hours
prior as previously described, for 30 or 60 minutes before washing off unbound virus and
replacing the EMEM containing 2.5% FBS and 1% PS and no RBD or control proteins. The
cells were fixed, and fluorescence was detected (Appendix 11). Cell confluency was detected
using brightfield microscopy (Appendix 12). Prolonged incubation of RSV at 37oC has been
shown to reduce viability of the viral particle58.
2.5 RSV F triggering assay
Spontaneous triggering of pre-fusion RSV F on the viral particle has been shown by Killikelly et
al58. However, during the fusion process, the triggering of RSV F on the host membrane, and
the role of nucleolin has yet to be characterised. By using both D25 and AM14 the associating
orientation of RBD1,2 with viral envelope pre-fusion RSV F as well as possible triggering was
analysed. D25 and AM14 are two pre-fusion specific antibodies which bind different epitopes. If
RBD1,2 blocks one epitope but does not induce triggering, the other antibody can still detect
the pre-fusion protein. Concentrated virus (105 FFU/mL) in EMEM, with 5% FBS and 1%PS
was incubated with 1μM RBD1,2 or just the described media for 2 hours and dot blotted onto a
nitrocellulose membrane at decreasing 3-fold concentrations. Protein was visualized using the
same blocking and antibody incubations as previously described for Western blots.
30
Chapter Three: Results
31
3.1 Expression and Purification of Proteins
RBD1, RBD2, and RBD1,2
Overall the E.coli expression system was robust and produced 10-20mg of RBD1,2 per litre of
media. RBD1,2 migrated to approximately 22kDa and RBD1 and RBD2 migrated to 10kDa on a
SDS-PAGE gel without boiling (Figure 5A and 5B). RBD1,2 and RBD1 were detected using
H250 polyclonal, as well as MS-3 monoclonal anti-nucleolin antibody. RBD2 was detected
using H250 polyclonal anti-nucleolin antibody.
Figure 5. RBD expression and purification. A) Western blot of SDS-PAGE and B) Coomassie SDS-PAGE. RBD1 in lanes 1, 2, and RBD2 in lane 3 migrating to their expected sizes (10kDa) relative to RBD1,2 at 22kDa (Lane 4). A heavier species in RBD2 (~15 kDa) was also seen with RBD2, detected by anti-HisTag and H250 anti-nucleolin polyclonal antibody. RBD samples were diluted in SDS-2-mercaptoethanol without boiling to prevent aggregation of RBD1.
SEC showed a single peak eluting at approximately 22kDa and 10kDa for RBD1,2, and RBD1
and RBD2 respectively (Figure 6A, 6B, and 6C).
32
33
Figure 6. RBD Size Exclusion Chromatography. Elution diagrams showing elution peaks at 37mL (A), 39mL (B), and 39mL (C) corresponding to 22kDa, 10kDa, and 10kDa.
Aggregation of RBD1 in solution and on SDS-PAGE was initially a concern. RBD1 aggregated
at concentrations over 1.5 mg after storage of purified proteins at 4oC for three days in storage
buffers previously described. This was prevented by lowering the concentration. Aggregation
was also witnessed when dialysing the purified protein in TBS, and PBS, this effect was
immediate and white precipitate formed on the dialysis membrane. The removal of imidazole
was identified as the cause of this and varying concentrations of imidazole were tested to
prevent aggregation. Imidazole is a highly polar compound with strong positive charges
especially when protonated and the effect of imidazole on His-tagged protein solubility has
been previously studied59. Secondly RBD1 protein aggregated in the well of SDS-PAGE. This
effect is witnessed in a variety of hydrophobic proteins60 and I observed RBD1 would not
aggregate in SDS-PAGE if the purified protein was not boiled in loading buffer prior to SDS-
PAGE.
34
RBD2 expression resulted in a readthrough product, confirmed by protein mass-
spectrophotometry, elongating the protein by 20 amino acids past the stop codon, resulting in a
second band approximately 2 kDa larger on SDS-PAGE (Figure 5A and 5B). The second band
was present at approximately ⅓ of the concentration of the expected band. Sequencing the
plasmid preparation did not identify any mutations that may cause this. The second band
remained despite stringent purification on nickel columns, and can bind RBD2 binding
antibodies as well as our anti-HisTag antibody. The heavier band could not be detected with
RBD1 or RBD1,2 specific antibodies. We sent the band, isolated from SDS-PAGE, for mass
spectrometry, and identified translated regions downstream of the stop codon (Appendix 13).
Readthrough products occur abundantly in nature and have a variety of functions61-63. The
cause of the readthrough products vary from protein interaction to secondary nucleic acid
structures. It is also possible a mutation in the stop codon occurred during plasmid preparation
and cloning. If a copy of the mutant plasmid and the correct plasmid was transformed into a
single E.coli and was selected as the most abundant expressing colony during protein
expression optimization it would have been retained as the stock culture for RBD2.
Furthermore due to the different abundances of both mutant and desired RBD2 sequences in
the bacteria, sequencing the plasmid did not give any clues as to the phenomena. Going back
to the plasmid preparation for RBD2 and by transforming new competent expression E.coli we
can eliminate the possibility that it was a mutation, but rather a true readthrough product, or
eliminate the heavier band by selecting for a well-expressing RBD2 without the heavier band.
RBD1,2 was expressed and purified successfully as previously described. RBD1,2, sharing
100% sequence identity of RBD2 and HisTag, purified samples also occasionally showed a
heavier band approximately 2kDa heavier than the dominant band. Selection of the RBD1,2
expressing transformed E.coli was performed by a student prior to my research project, thus
this does not support nor refute the possibility of either the possibility of a stop codon mutation
or some form of readthrough. On the other hand, boiling of RBD1,2 in SDS does not cause
35
extensive aggregation as seen in RBD1, although, in high concentrations of protein, boiling in
SDS loading buffer caused artefactual dimerization. Potential shielding of aggregation sites by
the residues on the remainder of the protein is the most likely cause. Another student in our lab
employed EMSA to visualize the RNA binding capacity of RBD1,2. Using sensitive fluorescent
labelling he was not able to detect RNA contamination from protein expression and purification
and identified the reduced binding capacity of urea purified RBD1,2 as well as the reduction in
the binding capacity over time. This indicates that RBD1,2 is only stable for a limited amount of
time in the storage conditions previously described. Optimization of the storage conditions,
including storage in -20oC or -80oC with or without a variety of cryopreservants, can be
conducted for long term storage of the protein, indicated by loss of native binding capacity to
NRE.
Pre- and Post-Fusion RSV F
The RSV F proteins expressed well in the media and were purified using both nickel and
streptactin columns (Figure 7A) and successfully cleaved using recombinant thrombin as
previously described. RSV F was further purified and stored at 4oC. Pre-fusion RSV F in SDS
with and without boiling in loading buffer loses trimerization and migrates to 60 kDa. When a
high concentration of pre-fusion F is loaded rapidly onto the gel without boiling, a small portion
of the protein remains trimerized and migrates to 180 kDa. Post-fusion F migrates to 50 kDa
when boiled in loading buffer, losing trimerization and the F2 peptide which is held on to F1 by a
disulphide bond. Without boiling the post-fusion F protein is capable of maintaining trimerization
in loading buffer for up to 12 hours at 4oC but still loses the F1 peptides migrating to 150 kDa.
Palivizumab was the most sensitive antibody for detecting our recombinant RSV F proteins,
followed by anti-HisTag and anti-StrepTag antibodies in that order.
36
Figure 7. RSV F expression and purification. A) Silver stained SDS-PAGE gel where the samples were boiled in SDS-2-Mercaptoethanol for 5 min. Lanes 1 and 2 showing Nickel column flow through and wash respectively. Lanes 3 and 4 showing column eluates of pre-fusion and post-fusion harvest respectively. Pre-fusion has F1 and F2 in a single chain at 60 kDa (lane 3 single band) whereas reducing conditions of gel separated F1 (lane 4 top band) and F2 (lane 4 bottom band) of post-fusion 50kDa and 10kDa respectively. B) Pre-fusion and post-fusion dot blot. Lanes 1 and 2, 10 ug pre- and post-fusion RSV F were dot blotted on a nitrocellulose membrane respectively. Palivizumab was used to detect both conformations in row 1. Rows 2 and 3 were detected using AM14 and D25, respectively, showing only the pre-fusion conformation. AM14 was likely a more sensitive antibody given the presence of three epitopes per RSV F trimer, whereas only one D25 epitope is accessible per trimer.
The RSV F proteins were capable of forming stable trimers, visualised using blue native-PAGE
(Figure 8A). Both RSV F Proteins eluted at approximately 180kDa (Figure 8B) However, the
trimers multimerized into triplets over time, with a significant portion by approximately one
month, as previously characterized10 (Figure 8C).
37
Figure 8. RSV F trimerization and size exclusion chromatography (SEC). A) Blue Native-PAGE gel showing bands at 180 kDa. Post-fusion RSV F sample over time also showed a band at 540 kDa. B,C) SEC elution diagrams showing UV absorbance peaks at 27 mL elution fraction corresponding to 180 kDa for both conformations and at 23 mL elution fraction corresponding to 540 kDa multimer for post-fusion RSV F.
A C-terminal fibritin domain must be added to the expressed ectodomain pre-fusion RSV F to
maintain the trimerized state. This suggests viral particle surface pre-fusion trimerization is less
stable than the post-fusion conformation. On the other hand, the pre-fusion protein does not
have as many exposed hydrophobic surfaces and it does not form the multimers of trimers that
post-fusion does, previously described as rosette formations. Post-fusion RSV F remains
38
predominantly trimers for two weeks as indicated by time course sample analysis on the SEC.
Pre- and post-fusion RSV F proteins were stored in TBS and PBS solutions at 4oC.
Optimization of the storage conditions, including storage in -20oC or -80oC with or without a
variety of cryopreservants, can be conducted for long term storage of the proteins, indicated by
the ratio of pre- to post-fusion RSV F in the pre-fusion RSV F sample and the rate of rosette
formation in post-fusion RSV F sample. Pre-fusion conformation was verified by two different
pre-fusion antibodies although both antibodies detected less overall fusion protein in the
samples than Palivizumab. There is the possibility that there is a portion of pre-fusion protein
that has switched to the post-fusion conformation, which has been previously described.
Luckily the exposure of the fusion peptide would rapidly cause aggregation of conformation
switched pre-fusion and sample can be purified of aggregates by centrifugation at 30 000 x g. It
is also possible Palivizumab has a higher affinity to pre-fusion and thus a stronger signal is
detected after the washes during the dot blots.
Pre-Fusion Specific Antibody
We detected pre-fusion RSV F in our pre-fusion sample, and we detected no pre-fusion in our
post-fusion sample (Figure 7B). Though we could not determine the proportion of pre- to post-
fusion RSV F in our pre-fusion samples, if our pre-fusion protein were to switch conformations
into post-fusion, it would expose the fusion peptide and cause aggregation and precipitation.
Post-fusion RSV F in our pre-fusion purification, therefore, could be removed from our pre-
fusion samples.
Size exclusion chromatography
Nickel purified RBD1, RBD2, and RBD1,2 (Figure 6A, 6B, and 6C) and StrepTactin and nickel
columns purified pre- and post-fusion RSV F (Figure 8B and 8C) were run on Superdex 200
SEC columns. RBD1,2 eluted as a single peak at approximately 22 kDa (Figure 6A) and RBD1
(Figure 6B) and RBD2 eluted at approximately 10kDa (Figure 6C). All the proteins ran
39
according to their expected sizes. Pre and post-fusion RSV F both showed elution peaks at
approximately 180 kDa (Figure 8B and 8C), though the previously described 540 kDa multimer
peak of post-fusion was also present (Figure 8C).
SEC pre-fusion RSV F and RBD1,2 co-elution
A peak eluted at greater than 600 kDa (Figure 9A) and was run on SDS-PAGE and blotted for
both RSV F and RBD1,2 (Figure 9B). The eluted fraction contained both RBD1,2 and RSV F.
RSV F runs at 180 kDa and RBD1,2 runs at 22 kDa, however the combined size was over 600
kDa. This can indicate that the binding does not occur at a single site and the possible
formation of a larger complex, or an artefactual aggregate of RBD1,2 and RSV F is present.
Repeating the experiment in the same conditions did not yield the same results.
40
Figure 9. Co-elution of RBD1,2 and pre-fusion RSV F on size exclusion chromatography (SEC). A) SEC elution diagram showing UV absorbance peaks at 19, 27, and 37 mL elution fractions corresponding to >600, 180, and 22 kDa respectively. B) SDS-PAGE and western blot of each elution fraction from SEC. Blotted with Anti-His-tag antibody to visualize for RBD1,2 and pre-fusion RSV F.
41
3.2 Co-precipitation of RBD1,2 and RSV F
RBD1,2 Co-precipitation with RSV F Precipitation
The StrepTactin Beads were able to precipitate both conformations of RSV F (Figure 10A)
A
B
Figure 10. Immobilized RSV F co-precipitates RBD. After incubating the input material and performing the co-precipitation with the StrepTactin beads, the unbound material was washed away, the eluates were run on a SDS-PAGE gel and blotted using Palivizumab to detect RSV F (A) and H250 rabbit polyclonal anti-Nucleolin antibodies to detect RBD (B). Pre- and post-fusion RSV F (bait) incubated without RBD, and RBD1, RBD2, and RBD12 (analyte) was incubated without RSV F were run as controls. NRE was added to RBD1,2 to elucidate its effect on binding. Post-fusion RSV F with SDS-2-mercaptoethonal without boiling migrated as a trimerized protein at 150kDa, losing all three F2 domains, whereas pre-fusion RSV F migrated as a monomer consisting of both F1 and F2 at 60kDa and as a trimer at 180kDa.
Both conformations of RSV F were able to co-precipitate RBD1,2 (Figure 10B). There was no
noticeable difference between the amount of RBD1,2 pulled down by pre- and post-fusion RSV
F, and no consistent difference with the addition of NRE. Overall, this suggests there is an
interaction between RBD1,2 with pre- and post-fusion RSV F, which has never been described
before. Pre-fusion RSV F was not able to co-precipitate RBD1 or RBD2, but post-fusion RSV F
was occasionally able to co-precipitate minimal amounts of RBD1.
42
Post-fusion RSV F had co-precipitated RBD1 but the co-precipitation was difficult to reproduce
reliably when post-fusion RSV F was immobilized. This may be due to a lower affinity. This
suggests both RBD1, and RBD2 is needed in close proximity to interact with pre-fusion RSV F
and the interaction of RBD1,2 with post-fusion RSV F is stronger than when it is RBD1 alone. A
combination of possibilities exist to support these results. RBD1 may have a majority of the
interacting residues, predominantly accessible on the post-fusion conformation, but require
RBD2 to stabilize the interaction, otherwise the affinity is low. On the other hand it is possible
the major interacting residues between pre- and post-fusion RSV F with RBDs is different
altogether, RBD1 having greater importance in binding with post-fusion whereas RBD1,2 is
required for binding with pre-fusion.
RSV F Pull Down with RBD1,2
The RBD proteins or milk was crosslinked to the NHS beads and RSV F proteins were
incubated with the beads generated (Figure 11).
43
A
B
Figure 11. Input material for RBD immobilization and RSV F precipitation. A) Input materials for the NHS binding and (B) RSV F pull-down was run on a SDS-PAGE gel and blotted using an H250 and Palivizumab respectively. Milk bound beads incubated with a mixture of pre- and post-fusion RSV F (lane 1), and RBD1, RBD2, and RBD1,2 without RSV F was run as control. RBD1,2 bound beads incubated with an equimolar solution of pre- and post-fusion RSV F. Post-fusion RSV F with SDS-2-mercaptoethonal without boiling migrated as a trimerized protein at 150kDa, losing all three F2 domains, whereas the majority of pre-fusion RSV F migrated as a monomer consisting of both F1 and F2 at 60kDa. There however was a small fraction of pre-fusion RSV F that ran as a trimer at 180kDa.
The RBD1,2 bound beads were successfully able to pull down both pre- and post-fusion RSV F
while the milk bound beads and RBD2 beads did not (Figure 12). RBD1,2 bound beads
preferentially pulled down pre-fusion RSV F from the equimolar solution of both pre- and post-
fusion RSV F when incubation and washing was performed without detergent (Figure 12A).
However, when the incubation and washes are performed using 0.01% Tween20, post-fusion
RSV F binds RBD1, and RBD1,2 better (Figure 12B).
44
A
B
Figure 12. Immobilized RBD precipitates RSV F. RSV F was incubated with RBD bound NHS beads in saline (A) or in saline supplemented with 0.01% Tween-20 (B). Milk bound beads incubated with a mixture of pre- and post-fusion F (lane 1), and RBD1, RBD2, and RBD1,2 without RSV F in saline or in saline supplemented with 0.01% Tween-20 was run as control. RBD1,2 bound beads incubated with an equimolar solution of pre- and post-fusion RSV F. Post-fusion RSV F with SDS-2-mercaptoethonal without boiling migrated as a trimerized protein at 150kDa, losing all three F2 domains, whereas the majority of pre-fusion RSV F migrated as a monomer consisting of both F1 and F2 at 60kDa. Washing away the unbound material with saline or with saline supplemented with 0.01% Tween-20, the eluates were run on a SDS-PAGE gel and blotted using Palivizumab to detect RSV F.
This indicates possible competition between pre- and post-fusion RSV F while favoring the
binding of one conformation over another depending on the incubation conditions, or different
interacting residues between the different RSV F conformations with RBD proteins, which are
independently improved or inhibited by the presence of detergents in the solvent. Besides
protein-protein interaction, the detergent may impact the solubility or the charges on the protein
themselves, altering the interaction between the two. Identifying a robust method of measuring
the dissociation constants between the RSV F protein and the RBD proteins would allow for
identification of the exact residues that are interacting. This may reveal the reason behind this
result. RBD1 was able to pull down post-fusion RSV F as well, but to a lesser degree (Figure
12). The reciprocal co-precipitation suggests RBD1,2 may bind RSV F at 1:3 ratio given that
RSV F naturally exists in a trimerized form and higher quantities of RSV F is eluted from the
45
RBD1,2 beads than RBD1,2 from the RSV F precipitation using Streptactin beads in the
previous co-precipitation experiments (Figure 10 and12).
Viral Particle Co-precipitation with RBD1,2 Pull Down
The RSV particles were co-precipitated using RBD1,2 (Figure 13). Co-precipitation of RBD1,2
with recombinant RSV F sheds light into the interaction between nucleolin and RSV F.
Whereas this co-precipitation is evidence suggesting our recombinant model is translatable to
the viral cell culture model. The reciprocal, showing recombinant RSV F is able to interact with
cell surface nucleolin, would similarly strengthen our recombinant models.
46
Figure 13. Immobilized RBD1,2 co-precipitates RSV A2 virus particles. A) Input materials for the virus particle co-precipitation using RBD1,2 (22kDa) as the bait was run on SDS-PAGE and detected using H250 anti-nucleolin polyclonal and RSV3 (detects RSV nucleoprotein 45kDa) anti-RSV triple monoclonal antibodies. Lane 1 RBD1,2 was incubated with virus, lane 2 RBD1,2 was incubated with FBS, and lane 3 RSV was incubated with BSA, before the samples were pulled down using Nickel beads. B,C) The same samples were eluted after washing and run on SDS-PAGE in the same order. The eluates were detected using either H250 (B) or RSV3 (C). RSV was successfully co-precipitated by RBD1,2.
3.3 Biolayer interferometry
In a wide variety of buffers and test conditions (Appendix 6), we discovered no binding was
detectable using the BLItz system (Figure 14). The co-precipitation interaction was conducted
at room temperature for two hours whereas the BLItz interaction was limited to a shorter period
of exposure. Loading RSV F on Steptactin Biosensors (Figure 14A and 14B) had shown a shift
in wavelength but binding was not detected. Similarly Loading for RBD1,2 onto anti-pentaHis
biosensors was successful without binding in the association phase (Figure 14C). Antibody
bind to RBD1,2 was used as a positive control.
47
Figure 14. Biophysical interaction using biolayer interferometry.
A
B
48
C
Figure 14. Biophysical interaction using biolayer interferometry. A) RBD1,2 was loaded onto Anti-Penta-His biosensors and subsequent analytes were added. B,C) Pre-fusion (B) and post-fusion (C) RSV F was loaded onto Streptavidin biosensors and subsequent analytes were added.
3.4 Cell Culture effects of RBD on RSV infection
RBD protein effects on cell metabolic activity
Although RBD1 and RBD1,2 decreased HEp-2 metabolic activity in the high nM range (Figure
15), neither protein had any effect in the tested low nM and pM concentration range. Neither
RBD2 nor Palivizumab had any impact on metabolic activity. Each concentration was tested at
least in triplicate per experiment. Error bars represent average standard error over the number
of replicates of the experiment performed at each concentration.
49
Figure 15. RBD toxicity assay. RBD and control proteins were added to HEp-2 cells with concentrated RSV A2 virus in EMEM with 2.5%FBS and 1%PS. Unbound viral particles were washed off using saline and fresh EMEM mixture without RBD and control proteins were added to the cells. Cell metabolic activity was measured after 24 hours using MTT assay.
Fluorescence quantification of RSV A2 infection
Raw fluorescence units were measured using various dilutions of RSV A2 samples and an
acceptable dilution was selected. Each concentration was performed in replicates of six and
error bars represent standard error of the experiment (Figure 16).
50
Figure 16. RSV fluorescence quantification. RSV was added to HEp-2 cells at varying dilutions to test fluorescence intensity of concentrated and fresh virus preparations for inhibition assay. Unbound virus was washed off using saline at 1 hour after incubation of the virus with the cells.
Infection inhibition with RBD
RBD1,2 and Palivizumab had successfully inhibited infection in the low nanomolar range in the
repeated experiment. RBD1 had also shown a 40% reduction in infection at 20nM whereas
RBD2 had no effect on viral infection (Figure 17 and 18). Each concentration was performed in
replicates of three and error bars represent standard error of the experiment. RBD1 and RBD2
was tested for RSV inhibition qualities using the same method previously described.
51
Figure 17. Cell culture RSV infection inhibition assay 24hr. RBD and control proteins were added to HEp-2 cells with concentrated RSV A2 virus in EMEM with 2.5%FBS and 1%PS. Unbound viral particles were washed off using saline and fresh EMEM mixture with RBD and control proteins were added to the cell. The cells were fixed at 24 hours and virus was detected using NCL-RSV3 antibody coupled with Alexa 488 fluorescent secondary antibody.
52
Figure 18. Cell culture RSV infection inhibition assay microscopy 24hr. RSV A2 incubated with 20nM of RBD or control proteins in EMEM with 2.5%FBS and 1%PS. Unbound viral particles were washed off using saline and fresh EMEM mixture with RBD and control proteins were added to the cells. The cells were fixed using at 24 hours and virus was detected using NCL-RSV3 antibody coupled with Alexa 488 fluorescent secondary antibody.
53
A wider range of concentrations can be tested for using the same protocol to display the
inhibition limits of RBD1. Evidence in the co-precipitation experiments show that RBD1
interacts with post-fusion RSV F with a low affinity. This interaction may be responsible for the
inhibitory effect through steric hindrance of the virus particles. The abundance of post-fusion
RSV F on the viral particle makes it a more susceptible target for protein interaction.
Association of hydrophobic domains between RBD1 and post-fusion RSV F may also have a
solubility effect on the virus particles, making the virus particle less likely to precipitate and bind
to the cell surface in the allotted infection time. This may be tested for by using a greater range
of infection times and comparing the infection rates to control infections without RBD1. If the
inhibition is rescued relative to the control infections at longer infection times, it is indicative of
RBD affecting virus particle solubility as the more likely cause of the RBD1 inhibition. If there is
no change in inhibition then steric hindrance is the more favored mechanism. The same
experiment for RBD1 using longer infection times, can be applied to RBD1,2 to test its
mechanisms of inhibition.
It was expected that the longest exposure at 37oC prior to infection will have the greatest
relative effect of inhibition even without the addition of RBD after infection. We hypothesized
that RBD1,2 inhibits viral infection through the triggering of the pre-fusion RSV F and not
through competitive binding. However, no inhibition was detected at the tested concentrations
when fluorescence values were normalized by their relative metabolic activity changes caused
by RBD1 and RBD1,2 (Figure 19).
54
Figure 19. Cell culture RSV fusion inhibition assay. RSV A2 incubated with 200nM of RBD or control proteins in EMEM with 2.5%FBS and 1%PS. Unbound viral particles were washed off using saline and fresh EMEM mixture without RBD and control proteins were added to the cells. The cells were fixed using 1:1 methanol/acetone at 24 hours and virus was detected using RSV3 antibody coupled with Alexa 488 fluorescent secondary antibody.
These inhibition results suggest that RBD1,2 and RBD1 concentrations must be maintained
throughout the 24 hour propagation in order to prevent infection. It is possible that RBD1,2 and
RBD1 is sterically inhibiting the interaction of RSV F with cell surface nucleolin.
An unaccounted for variable in the protocol for our cell culture infection inhibition experiments is
the possibility of internalization of RBD proteins. Classically, fusion inhibition experiments
compare the ratio of infected cells to uninfected cells under treatment conditions. Our assay
instead looks at mean fluorescence quantified abundance of RSV proteins. If association of
RBD1 or RBD1,2 with RSV F affects other processes such as replication, RNA processing, or
other host dependent viral processes, the mean fluorescence would be decreased due to lower
abundance of viral proteins, independent of the fusion process. Confirmation of fusion inhibition
must be quantified using an FFU decrease to ensure it is an entry process that is inhibited.
Furthermore although unlikely, the attachment protein RSV G may be affected by RBD1 or
55
RBD1,2 as well. Experiments showing RSV G does not interact with RBD proteins can support
the hypothesis that RBD inhibits infection through the RSV F protein. Similarly inhibition
experiments can be performed using a RSV mutant virus without the attachment protein,
showing that the inhibition persists without the RSV G protein.
3.5 RSV F triggering assay
No significant decrease in pre-fusion RSV F was detected compared to the palivizumab loading
control (Figure 20). This is suggestive of RBD1,2 not triggering the viral particle pre-fusion F,
however this experiment should be replicated with purified virus and carried out in PBS.
56
Figure 20. RSV virus pre-fusion antibody binding. A,B,C) RSV A2 concentrated virus incubated with 1μM BSA in lane 1 and RBD1,2 in lane 2. Palivizumab (A) is a commercial anti-RSVF antibody detecting both pre- and post-fusion conformations of RSV F. D25 (B) and AM14 (C) are pre-fusion specific antibodies targeting an axial epitope site 0 and a lateral epitope respectively. Three fold dilutions were dot blotted onto a nitrocellulose membrane.
3.6 NRE Binding to RBD1,2
The effect of RNA binding on the interaction between RDB1,2 and RSV F proteins has not
been previously explored. Physiologically nucleolin has been shown to bind a range of RNA
targets, however the binding of NRE while on the cell surface has not been studied. These
experiments hoped to elucidate the state of nucleolin on the cell surface and the impact of NRE
binding on the RSV F interaction and its impact on the fusion process. If NRE binding is found
to play a role in the interaction, these observations will not only reveal previously unknown
57
information about the infection process, they can also introduce variety of treatment options
that target or supplement the binding of RNA to surface nucleolin. Another student from our lab
has confirmed recombinant RBD1,2 can recognize and bind the NRE. However the co-
precipitation results do not suggest a major impact of NRE binding on the interaction (Figure
10B). A more robust system for measuring quantitative biophysical interaction between the
proteins can verify the effect of NRE binding to RSV F.
58
Chapter Four: Discussion
59
4.1 Conclusions
My body of work aimed to shed light on both the biophysical and cell surface interaction
between RBD and RSV F. By implementing the use of recombinant proteins I can control more
variables in the interaction and identify the orientation of the two proteins. The exact peptides
responsible for interaction with the pre-fusion RSV F can be elucidated. Taking the knowledge
gained from the biophysical interaction studies, I attempted to complement the results obtained
in the cell infection inhibition models, and develop a clear model for the mechanism of the
fusion process, while encouraging the design of inhibitory peptides for potential therapeutic
use.
My project has identified interacting domains between nucleolin and RSV F and observed
differences between pre-fusion and post-fusion RSV F’s interaction characteristics. This
evidence sheds light onto both the pathogenesis process of RSV, as well as the possible
orientation of nucleolin on the cell surface. Better understanding of the both the host and the
virus gives direction for future research in characterisation of the important fusion process.
4.2 Summary
The work carried out in this thesis consists of numerous experiments that bring new knowledge
to the area of the interaction between different conformations of the RSV fusion protein and its
cellular receptor, nucleolin (as studied through two RNA binding domains, complexed together
and separately).
I was able to optimize the expression and purification of RBD1, RBD2, and RBD1,2 using an
E.coli system. Simultaneously, I optimized the expression and purification of pre- and post-
fusion RSV F in a HEK293T mammalian transfection system. The RBD proteins were
monomeric and the RSV F proteins were trimeric, and their storage conditions were assessed.
60
I established the novel interaction between RBDs and the RSV F proteins using reciprocal co-
precipitation of pre- and post-fusion RSV F with RBD1,2 identifying evidence suggesting that
RBD1 interacts with post-fusion RSV F as well. I showed that RBD1,2 preferentially binds pre-
fusion RSV F in detergent free conditions, whereas post-fusion F has higher affinity to RBD1,2
with addition detergent. Adding detergent also increased the amount of post-fusion RSV F co-
precipitated by RBD1. I supplemented this result, by showing recombinant RBD1,2 can also co-
precipitate RSV A2 viral particles. My experiments have shown that the affinity of this
interaction between recombinant RBD1,2 and RSV F proteins cannot be quantified using BLI.
I have confirmed that RBD1,2 can inhibit RSV A2 infection of HEp-2 cells, when preincubated
with viral particles prior to infection, while maintaining RBD1,2 concentration during the
subsequent 24hour incubation period. I have shown that RBD1 can also inhibit viral infection
under the same conditions to a lesser extent than RBD1,2. I have observed that RBD proteins
can not reduce viral infection with preincubation prior to infection alone without maintaining
RBD concentrations in the subsequent 24hour incubation period. This observation is
supplemented by the fact that pre-fusion conformation of F protein on RSV A2 viral particles
does not decrease in relative abundance when incubated with RBD1,2 compared to controls.
4.3 Cell Surface nucleolin
The orientation of nucleolin on the cell surface has never been studied. My experimental results
support the possibility that the central regions of nucleolin are exposed to the extracellular
lumen, firstly suggesting its physical orientation allows for the exposure and access of RBD1,2
to RSV F during infection, and secondly the results support the hypothesis that cell surface
nucleolin has a role in binding extracellular nucleic acid targets considering the exposed RBDs.
Adding to the diverse functions and interactions, this can be yet another function of nucleolin in
61
addition to those previously described. AS1411 is a DNA aptamer originally described to bind
cell surface nucleolin in cancer cells, which overexpress the protein, as a cancer-cell targeting
mechanism64. This hypothesis is further supported by reports of AS1411 having anti-viral
effects in HIV infections. It has been suggested that AS1411 binding of cell surface nucleolin
induces its internalization and reduces the overall amount of cell surface nucleolin available65.
This effect is further explored in a recent publication describing the use of AS1411 as an anti-
viral therapeutic option for treating RSV infection57, potentially through the same mechanism,
by making the cellular receptor unavailable for infection. Nuclear-cytoplasmic transport for pre-
ribosomal subunits has been identified as a function of nucleolin. However, cytoplasmic
nucleolin either get rapidly transported back into the nucleus, or degraded. Under specific
stress conditions nucleolin has been shown to leave the nucleus43,44. However the ability for cell
surface nucleolin to auto-regulate the localization or the expression levels have never been
described. These results indicate the potential role of nucleolin as a receptor to extracellular
nucleic acid signals. However more work needs to be done to support this hypothesis.
4.4 RSV Pathogenesis
Concerning RSV pathogenesis, our results support the RSV interacts with nucleolin as reported
by Tayyari et al.2 and we have evidence that both conformations RSV F can interact with
nucleolin through RBD1,2. This interaction may be a crucial step in understanding the fusion
process. After RSV viral particles are anchored to the cell surface by RSV G to one of its many
attachment receptors as previously shown in literature9,10, RSV F can then interact with RBD1,2
domains of cell surface nucleolin during the fusion process, which has never been shown
before (Figure 21). Our cell culture results indicate that preincubation of recombinant RBD1,2
with the viral particle is not sufficient in causing a permanent inhibition of RSV A2, suggesting
that pre-fusion RSV F, although is interacting with RBD1,2, is not changing conformation
prematurely and thus permanently losing function. Coupled with the inhibition capability of RBD
62
proteins when the concentrations are maintained for the 24 hours post-infection, I hypothesize
that recombinant RBD proteins in solution transiently associate with RSV F domains as long as
concentrations are maintained, resulting in steric hindrance and the loss of RSV F’s ability to
interact with cell surface nucleolin and induce fusion. This suggests that the cell surface
RBD1,2 interaction with RSV F may only be one of the steps involved for the fusion process
(Figure 21B). These results indicate other potential triggers for pre-fusion conformation change.
Candidates for the trigger include a different domain of cell surface nucleolin, other lipid raft
proteins associated with nucleolin, and even the absence of a well defined trigger is possible.
Pre-fusion RSV F has been shown to spontaneously trigger into the post-fusion conformation
on the viral envelope58, and the binding of the nucleolin receptor may be facilitating the
spontaneous triggering event to occur in close proximity to the host lipid membrane, inducing
fusion. This spontaneous triggering model, which utilizes nucleolin as the guidance receptor,
would support only a transient interaction between nucleolin and the viral fusion protein. Tight
association would hinder viral particle budding later in the life cycle of RSV infection.
63
Figure 21. Proposed RSV fusion model. A) RSV G attaches to surface receptors. B) RSV F interacts with RBD1,2 domains of nucleolin, potentially interacting with other regions of nucleolin, the lipid membrane, and/or lipid raft proteins. C) RSV F is triggered inducing membrane fusion.
64
4.5 Future Directions
Cell Surface Nucleolin and Recombinant RSV F
Co-precipitation results can be generated using cell surface purified nucleolin instead of
recombinant RBD1,2, while demonstrating preferential binding to the recombinant pre-fusion or
post-fusion RSV F. This will add strength to the cell model of the interaction. The reciprocal,
RBD1,2 and viral particles has already been performed. Furthermore competitive binding of cell
surface nucleolin and recombinant RBD1,2 can be performed. Results for the experiment may
support the new hypothesis that viral particle pre-fusion RSV F preferentially binds recombinant
RBD1,2 sterically preventing it from interacting with the cell surface receptor.
More in depth studies of cell surface nucleolin and its associated proteins can also shed light
on other potential candidates which may be triggering RSV F. The functions of cell surface
nucleolin can be further elucidated by analysing a variety of effectors of nucleolin
transportation. Inhibition of the factors which cell surface nucleolin is dependent on for
localization to the cell surface can effectively reduce cell surface nucleolin without effectively
killing the cell model.
Revisiting the Sf9 insect cell transfection model can be useful in determining the regions of
nucleolin involved in RSV infection. The use of various different recombinant nucleolin
constructs used to transfect the cells can show which, regions are mandatory to permit
infection.
Isothermal Calorimetry
The affinity of RSV F and RDB proteins can also be calculated using ITC. The RBD ligands can
be added to the RSV F proteins at a 10:1 molar ratio and the interaction will determine the
65
stoichiometry, enthalpy, and Kd. ITC should be run at its maximum sensitivity, approximately
500μM Kd, using 3.6mg of RSV F proteins at 100μM in the 200μL chamber and adding 4.5mg
of RBD ligands at 10mM in the 40μL syringe. Following initial experiments, Kd and
stoichiometry adjustments can be made. The initial run assumes a 3:1 RSV F to RBD or a
trimer to monomer stoichiometry at a low 500 μM Kd. This is hypothesized from fact that RSV F
co-precipitates less RBD1,2 than the reciprocal, RBD1,2 co-precipitating RSV F. A clear
understanding of the affinity RBD1,2 has for RSV F and a method of analysing the affinity will
be crucial in elucidating the functional role of the interaction. The affinities can be compared
between pre-fusion and post-fusion, as well as, between cell surface nucleolin and recombinant
RBD1,2.
Site Directed Mutagenesis
With a robust method to measure the interaction strength between the recombinant proteins,
site directed mutagenesis can be employed to identify key amino acids involved in the
interaction between RSV F and the RBD1,2. Using structure-based computational predictions
of protein-protein interactions, amino acids of interest can be mapped out. The modified
recombinant RBD1,2 proteins aims to identify which substitutions directly affect binding affinity.
Cell Culture Experiments
To further validate the mechanism by which RBD1,2 inhibits infection, the infectivity of RSV A2
live virus can be rescued using recombinant RSV F incubated with all the generated inhibitory
recombinant proteins prior to exposure to the viral particles. This demonstrates the binding of
the recombinant RSV F to the inhibitors reducing the binding to viral particles, thus rescuing
infectivity. It can be hypothesized that recombinant RSV F can bind a portion of the
recombinant RBD1,2, however, it is possibile that viral particle surface RSV F has greater
affinity to the RBDs. This will buffer the rescue effect depending on the relative Kd.
66
The inhibition of RSV A2 can be replicated using other RSV strains to ensure the effect is not
isolated with the A2 strain. Other common RSV laboratory strains are RSV Long and RSV BA.
67
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74
6. Appendix
Appendix 1. Amino acid sequences of RSV F from RSV A2, pre-fusion construct, and post-
fusion construct (generated using T-Coffee66).
HRSVA2F MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKC
PreFusion -------------------------QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKIKC
PostFusion -------------------------QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKC
cons ****************************************: **
HRSVA2F NGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFL
PreFusion NGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNQARGS----------------------GSGRSL
PostFusion NGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARR-----------------------------
cons ******:*************************.***:**
HRSVA2F GFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIV
PreFusion GFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIV
PostFusion --------AIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIV
cons *************************************************************
HRSVA2F NKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN
PreFusion NKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN
PostFusion NKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN
cons *******.*************************************************************
HRSVA2F NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDN
PreFusion NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDN
PostFusion NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDN
cons *********************************************************************
HRSVA2F AGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIV
PreFusion AGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIV
PostFusion AGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIV
cons *********************************:***********************************
HRSVA2F SCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVF
PreFusion SCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVF
PostFusion SCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVF
cons ********************************:************************************
HRSVA2F PSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAG-------------KSTTNIMITTIIIVIIVILL
PreFusion PSDQFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL--------
PostFusion PSDEFDASISQVNEKINQSLAFIRKSDELLGLE---------------------VLF---Q--------
cons ***:************************** -
HRSVA2F SLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN
PreFusion -----------------------------------
PostFusion -----------------------------------
cons END
BAD AVG GOOD
75
Appendix 2. Amino acid sequences of RBD1,2, RBD1, and RBD2 (generated using T-Coffee64).
RBD12 GTEPTTAFNLFVGNLNFNKSAPELKTGISDVFAKNDLAVVDVRIGMTRKFGYVDFESAEDLEKALELTGLKVFGN
RBD1 GTEPTTAFNLFVGNLNFNKSAPELKTGISDVFAKNDLAVVDVRIGMTRKFGYVDFESAEDLEKALELTGLKVFGN
RBD2 ---------------------------------------------------------------------------
RBD12 EIKLEKPKGKDSKKERDARTLLAKNLPYKVTQDELKEVFEDAAEIRLVSKDGKSKGIAYIEFKTEADAEKTFEEK
RBD1 EIKLEKPKG------------------------------------------------------------------
RBD2 ------------------------NLPYKVTQDELKEVFEDAAEIRLVSKDGKSKGIAYIEFKTEADAEKTFEEK
RBD12 QGTEIDGRSISLYYTGEPKGEGLEHHHHHH
RBD1 -----------------------HHHHHHH
RBD2 QGTEIDGRSISLYYTGEPKGEGLEHHHHHH
BAD AVG GOOD
76
Appendix 3 Antibodies
Antibodies Antibody type Target Reference/Catalogue Number
MS-3 Mouse IgG Monoclonal
Nucleolin sc-8031 (SantaCruz)
H250 Rabbit IgG Polyclonal
Nucleolin sc-13057 (SantaCruz)
Palivizumab Human IgG Monoclonal
RSV F Synagis
HisTag Monoclonal Antibody (100ug)
Mouse IgG Monoclonal
HisTag 70796-3 (Millipore)
NCL-RSV3 Mouse 3x IgG Monoclonal
RSV F, RSV N, RSV Phosphoprotein
NCL-RSV3 (Leica)
Goat anti-Human IgG 2o Antibody, HRP
Goat IgG Polyclonal
Human IgG (abcam)
Goat anti-Mouse IgG 2o Antibody, HRP
Goat IgG Polyclonal
Mouse IgG ab97023 (abcam)
Goat anti-Rabbit IgG 2o Antibody, HRP
Goat IgG Polyclonal
Rabbit IgG ab97051 (abcam)
Alexa Fluor 488 Goat Anti-Mouse IgG (H+L)
Goat IgG Polyclonal
Mouse IgG a11001 (Thermo)
77
Appendix 4. RBD1, RBD2, RBD1,2 plasmid maps
RBD1
78
RBD2
79
RBD1,2
80
Appendix 5. RSV F expression plasmids
Pre-fusion F
81
Post-fusion F
82
Appendix 6. BLItz conditions
Buffers Concentration Conditions
Blocking Agent
1. BSA 5, 2.5, 1, 0.5, 0.1%
2. Milk 1, 0.5, 0.1% Buffer
1. TBS 150, 300, 450mM
2. PBS 150, 300, 450mM Tween 20
1. 0.01%
2. 0.02% Imidazole
1. 1mM
2. 10mM
3. 50mM
4. 100mM
RSV F
1. 10μg/mL
2. 25μg/mL
3. 50μg/mL
4. 75μg/mL
5. 100μg/mL RBD1,2
1. 100μg/mL
2. 200μg/mL
3. 300μg/mL
4. 500μg/mL
5. 1mg/mL
StreptAvidin Biosensor Loading times Second Baseline times Association times Temperature Mixing Tube vs. Holder
Buffers Concentration Conditions
Blocking Agent
1. BSA 5, 2.5, 1, 0.5, 0.1%
2. Milk 1, 0.5, 0.1% Buffer
1. TBS 150, 300, 450mM
2. PBS 150, 300, 450mM Tween 20
1. 0.01%
2. 0.02% Imidazole
1. 1mM
2. 10mM
3. 50mM
4. 100mM
RBD1,2 +/- NRE
1. 5μg/mL
2. 10μg/mL
3. 25μg/mL
4. 50μg/mL
5. 75μg/mL Thrombin cleaved RSV F
1. 100μg/mL
2. 250μg/mL
3. 500μg/mL
4. 750μg/mL
5. 1000μg/mL H250 or MS3 anti-nucleolin
1. 100μg/mL
2. 200ug/mL
Anti-PentaHis Biosensor Loading times Second Baseline times Association times Temperature Mixing Tube vs. Holder
83
Appendix 7. RBD viability 24 hr incubation
RBD1 Viability Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5
200nM 0.4247 0.4046 0.4047 0.3923 0.4007
40nM 0.4657 0.4532 0.4377 0.4228 0.4263
8nM 0.5365 0.5203 0.4889 0.4947 0.4958
3.2nM 0.5946 0.5902 0.5368 0.527 0.5373
640pM 0.6042 0.5943 0.5576 0.562 0.5341
128pM 0.6192 0.6064 0.5788 0.5662 0.5588
0 0.6252 0.6138 0.5997 0.584 0.5726
RDB 2 Viability Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Repeat 6
200nM 0.7223 0.6962 0.7009 0.3067 0.5591 0.5558
40nM 0.7036 0.6563 0.6575 0.4812 0.5522 0.5881
8nM 0.7123 0.6604 0.6515 0.4331 0.5261 0.5588
3.2nM 0.696 0.6553 0.6266 0.4051 0.4986 0.5237
640pM 0.6912 0.6483 0.623 0.3914 0.492 0.5508
128pM 0.6911 0.6618 0.6471 0.3259 0.4919 0.5387
0 0.713 0.6794 0.665 0.4074 0.5095 0.5474
RBD1,2 Viability Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Repeat 6 Repeat 7
200nM 0.5453 0.5451 0.5438 0.5099 0.4952 0.4924 0.5323
40nM 0.6164 0.6235 0.6351 0.5807 0.5913 0.588 0.6019
8nM 0.6498 0.656 0.6367 0.6572 0.6244 0.6478 0.6127
3.2nM 0.6686 0.6866 0.6807 0.6596 0.6595 0.6715 0.6204
640pM 0.7061 0.7151 0.7413 0.7086 0.7023 0.7014 0.6386
128pM 0.6868 0.6761 0.6752 0.674 0.6795 0.6802 0.5678
0 0.7117 0.7057 0.7131 0.6915 0.707 0.6883 0.6614
84
Palivizumab Viability Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Repeat 6
200nM 0.7228 0.7421 0.709 0.7091 0.7507 0.3842
40nM 0.7581 0.7244 0.7097 0.6679 0.7212 0.3956
8nM 0.7409 0.7392 0.7186 0.6931 0.7307 0.4372
3.2nM 0.7208 0.7277 0.6904 0.6943 0.714 0.4461
640pM 0.6751 0.7276 0.722 0.7026 0.7052 0.4342
128pM 0.6471 0.6932 0.7081 0.6987 0.7031 0.4435
0 0.6333 0.6772 0.6809 0.7112 0.7034 0.4167
p values
RBD1 RBD2 RBD1,2 Palivizumab
200nM < 0.00001 >0.05 < 0.00001 >0.05
40nM < 0.00000 >0.05 < 0.00001 >0.05
8nM < 0.00001 >0.05 0.003862 >0.05
3.2nM 0.013121 >0.05 >0.05 >0.05
640pM >0.05 >0.05 >0.05 >0.05
128pM >0.05 >0.05 >0.05 >0.05
85
Appendix 8. Fluorescence assay
Concentrated virus Fresh Virus
10x 41306 39556 39899 38572 31443 32223
50x 35273 35397 33762 30392 30668 28474
250x 33238 28942 29707 26998 26661 26777
1250x 28476 29260 28165 23778 25070 24882
6250x 25690 26976 25678 25402 24525 23842
31250x 27547 27470 26949 26916 24671 25851
156250x 26943 27524 27307 26765 26371 27924
No virus 27974 28135 26012 26906 25271 25787
Appendix 9. 24 hr incubation inhibition
RBD1 RBD2 RBD12 Palivizumab
20nM 38505 37904 37144 36241 35781 37847 36139 36410 37674 37110 35995 36110
4nM 36140 37441 37538 35897 35970 37768 36167 37370 37290 36248 34724 36547
800pM 38110 38162 37067 37030 35910 36681 37332 36824 37436 36798 36506 37279
160pM 37235 39234 36772 36616 36650 37043 36982 36929 37033 36394 36680 36902
0 32542 31742 31378 32185 32502 32900 33222 32447 32403 32228 32325 33974
p values
RBD1 RBD2 RBD1,2 P
20nM 0.013291 0.042156 < 0.00001 < 0.00001
4nM >0.05 >0.05 < 0.00001 < 0.00001
800pM >0.05 >0.05 >0.05 >0.05
160pM >0.05 >0.05 >0.05 >0.05
86
Appendix 10. Cell confluency for 24 hr incubation.
87
Appendix 11. 2 hr incubation (Fluorescence Units)
RBD1 Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Repeat 6
200nM 37194 34849 34867 34566 38494 34655
40nM 38531 35673 35539 35556 37356 34543
8nM 38804 35190 34349 36009 37519 33349
1.6nM 38210 35097 33626 35676 37509 34455
0.32nM 38150 36355 33803 35887 39355 34003
0.128nM 39398 35317 33981 35543 36317 36981
0nM 37697 35799 34180 34988 37995 35180
-neg 32111 31342 31234 31849 31285 31318
RBD2 Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Repeat 6
200nM 35499 33775 32821 34665 33111 33998
40nM 35662 33654 32297 33786 34565 33897
8nM 35927 33618 31995 34677 32421 34356
1.6nM 36742 33393 33595 35772 31143 35776
0.32nM 36723 34711 33654 35881 33242 36546
0.128nM 36057 33629 33981 35547 33478 34332
0nM 34461 33410 34229 34667 33556 34432
-neg 29578 26567 30791 29987 27661 30238
RBD1,2 Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Repeat 6
200nM 33303 34432 33972 32982 33898 33786
40nM 33091 34939 33001 33541 34776 31764
8nM 33696 34729 33214 33445 34981 32451
1.6nM 34657 34351 31960 32967 33123 34429
0.32nM 38567 36303 32692 35543 35128 32987
0.128nM 37013 34338 32235 35232 35665 32112
0nM 34421 34972 33402 34112 35441 33212
-neg 31256 29314 29796 30411 30998 28971
88
Palivizumab Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Repeat 6
200nM 32298 32362 31444 30112 31678 32109
40nM 29960 34269 31433 30661 33459 31278
8nM 31420 35244 32343 30431 34568 33002
1.6nM 29394 35069 38309 30013 35187 37641
0.32nM 27779 35105 39487 29789 35467 37661
0.128nM 30115 39896 40032 31124 36993 38001
0nM 32316 39999 37822 34341 36767 37032
-neg 30576 30684 31567 29665 30901 30879
p values
RBD1 RBD2 RBD1,2 Palivizumab
200nM >0.05 >0.05 >0.05 2.20E-05
40nM >0.05 >0.05 >0.05 0.000231
8nM >0.05 >0.05 >0.05 0.003909
1.6nM >0.05 >0.05 >0.05 >0.05
0.32nM >0.05 >0.05 >0.05 >0.05
0.128nM >0.05 >0.05 >0.05 >0.05
89
Appendix 12. Cell confluency for 2 hr incubation.
90
Appendix 13. RBD2 Readthrough Product
...H H H H H H STOP D P A A N K A R K E A E L A A A T A E Q STOP
