1
Inefficient control of host gene expression by the 2009 pandemic 1
H1N1 influenza A virus NS1 protein 2
3
Benjamin G. Hale1, John Steel
1, Rafael A. Medina
1,3, Balaji Manicassamy
1,3, Jianqiang 4
Ye4, Danielle Hickman
4, Rong Hai
1, Mirco Schmolke
1, Anice C. Lowen
1, Daniel R. 5
Perez4 & Adolfo García-Sastre
1,2,3,* 6
7
1Department of Microbiology,
2Department of Medicine, Division of Infectious Diseases, 8
3Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, One 9
Gustave L. Levy Place, New York, NY 10029, USA. 4Department of Veterinary 10
Medicine, University of Maryland, College Park, MD 20742, USA. 11
12
*Corresponding author. Mailing address: Department of Microbiology, Mount Sinai 13
School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Phone: (212) 14
241-7769. Fax: (212) 534-1684. E-mail: [email protected]. 15
16
Running Title: 2009 Pandemic H1N1 NS1 Protein and CPSF30. 17
18
Words in abstract: 248. 19
Words in main text: 6,942. 20
Number of figures and tables: 9 figures + 1 table. 21
Number of references: 60. 22
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.00081-10 JVI Accepts, published online ahead of print on 5 May 2010
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
2
ABSTRACT 1
2
In 2009, a novel swine-origin H1N1 influenza A virus emerged. Here, we characterize the 3
multifunctional NS1 protein of this human pandemic virus in order to understand factors 4
that may contribute towards replication efficiency or pathogenicity. Although the 2009 5
H1N1 virus NS1 protein (2009/NS1) is an effective interferon-antagonist, we found that 6
this NS1 (unlike those of previous human-adapted influenza A viruses) is unable to block 7
general host gene expression in human or swine cells. This property could be restored in 8
2009/NS1 by substituting R108, E125 and G189 for residues corresponding to human 9
virus consensus. Mechanistically, these previously undescribed mutations acted by 10
increasing binding of 2009/NS1 to the cellular pre-mRNA processing protein CPSF30. A 11
recombinant 2009 H1N1 influenza A virus (A/California/04/09) expressing NS1 with 12
these gain-of-function substitutions was more efficient than wild-type at antagonizing 13
host innate immune responses in primary human epithelial cells. However, such 14
mutations had no significant effect on virus replication in either human or swine tissue-15
culture substrates. Surprisingly, in a mouse model of pathogenicity, the mutant virus 16
appeared to cause less morbidity, and was cleared faster, than wild-type. The mutant virus 17
also demonstrated reduced titers in the upper respiratory tracts of ferrets, however contact 18
and aerosol transmissibility of the virus were unaffected. Our data highlight a potential 19
human adaptation of NS1 that seems absent in ‘classically-derived’ swine-origin 20
influenza A viruses, including the 2009 H1N1 virus. We discuss the impact that natural 21
future gain of this NS1 function may have on the new pandemic virus in humans. 22
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
3
INTRODUCTION 1
2
In April 2009, an antigenically distinct swine-origin H1N1 influenza A virus was 3
detected in humans (1). This virus has since spread efficiently around the world, leading 4
to the declaration of a global pandemic by the World Health Organization on 11th
June 5
2009. Although infection with the virus is generally associated with a mild self-limiting 6
influenza-like disease in the majority of people, the young and those with certain 7
underlying conditions (including asthma, diabetes, heart/lung problems, morbid obesity, 8
and pregnancy) seem at greater risk of severe disease progression (26). Lack of 9
significant pre-existing immunity to the novel virus in those <60 years old (21, 35) may 10
account for the observation that younger people are more susceptible to this virus (26). 11
12
Sequence analysis of the 2009 pandemic H1N1 influenza A virus genome has failed to 13
identify any previously recognized virulence markers (13, 17, 24). Nevertheless, animal 14
studies have indicated that the 2009 H1N1 virus is slightly more pathogenic than 15
contemporary human seasonal H1N1 viruses (24, 34, 38). In ferrets, the pandemic H1N1 16
virus replicated to higher titers than seasonal H1N1 viruses in the upper respiratory tract 17
(24, 34, 38) and, unusually, could be detected deeper in the lungs (24, 38) and intestinal 18
tracts (34) of infected animals. In a mouse model, the 2009 H1N1 virus also replicated 19
more efficiently, and caused greater morbidity and mortality, than seasonal influenza 20
viruses (24, 34). Furthermore, mice infected with the 2009 H1N1 virus produced higher 21
levels of several pro-inflammatory cytokines and chemokines, including IFNγ, IL4, IL5, 22
IL12, MIP1α, MIP1β, and RANTES (24). Similar data for the 2009 H1N1 virus with 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
4
respect to its efficient replication and significant induction of cytokines (together with 1
severe lung pathology) have also been obtained from experimentally infected cynomolgus 2
macaques (24). As might be expected given its swine origin, the 2009 H1N1 virus 3
replicates well in pigs, although whether the virus causes significant disease in this host is 4
unclear (24, 30). In experimental infections of avian species the 2009 H1N1 virus is 5
unable to replicate efficiently or cause disease (2, 54). 6
7
Influenza A virus virulence is a polygenic trait. Multiple unidentified molecular 8
determinants are likely responsible for the ability of the 2009 H1N1 influenza A virus to
9
cause increased disease severity in certain host species. Here, we have focused on the 10
non-structural (NS1) protein of the 2009 H1N1 virus. NS1 performs a number of diverse 11
roles in infected cells [reviewed in (19)]. The best-described function of NS1 is its ability 12
to limit host-cell cytokine production [e.g. interferon (IFN)] during infection (12, 40, 55, 13
59). This is due to at least two independent NS1 activities (28): (i) the pre-transcriptional 14
inhibition of RIG-I [through sequestration of this RNA helicase and its activating ligand 15
(15, 36, 44, 45), or inhibition of TRIM25-mediated RIG-I ubiquitination (11)], and (ii) 16
the global post-transcriptional inhibition of CPSF30-mediated cellular pre-mRNA 17
processing (8, 39). Both of these activities appear to contribute to the replication 18
efficiency of human influenza A viruses, and both likely play roles in determining 19
virulence (8, 11, 12, 56). 20
21
There are recently documented examples of non-human adapted virus strains (both 22
laboratory and naturally-occurring) that encode NS1 proteins lacking the ability to inhibit 23
CPSF30 (22, 28, 56). The commonly used egg- and mouse- adapted human-derived 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
5
influenza virus A/Puerto Rico/8/34 (PR/34) NS1 protein has amino-acid substitutions at 1
residues 103 and 106, and is consequently unable to bind CPSF30 or block general gene 2
expression (22, 28). The NS1 proteins of two avian-derived influenza A viruses (the 3
highly-pathogenic A/Hong Kong/483/97 [H5N1] strain that was transmitted to humans in 4
1997, and its presumed precursor A/Teal/Hong Kong/W312/97 [H6N1]) are additionally 5
unable to inhibit CPSF30 in either human or avian cells, an intrinsic defect in NS1 6
function that also maps to substitutions at positions 103 and 106 (56). Sequence analysis 7
indicates that such substitutions are common amongst avian H6 and H9 HA subtypes, and 8
it has been suggested that these viruses may tolerate (or even be selected for) inefficient 9
CPSF30 binding (56). Furthermore, NS1:CPSF30-destabilizing substitutions at positions 10
103 or 106 have been reported to arise when viruses are adapted to replicate well in 11
certain new host species [e.g. duck to quail (23), and human to mouse (5)]. The biological 12
reasons and apparent selection pressures required for some viral strains to modulate their 13
NS1:CPSF30 binding affinities in particular hosts are unclear. 14
15
The ability of swine-derived influenza virus NS1 proteins to function efficiently in human 16
cells has not yet been fully tested. Galvanized by the worldwide spread of swine-origin 17
H1N1 influenza A virus in humans, we assessed the effectiveness of this pandemic virus 18
gene product to antagonize the human innate immune response. Surprisingly, we found 19
that the 2009/NS1 protein is unable to block general host gene expression in both human 20
and swine cells. This is a consequence of previously undescribed amino-acid changes in 21
NS1 that reduce its binding to CPSF30. Such changes appear to have occurred as soon as 22
the parental virus from which this NS1 is derived (likely the 1918 pandemic H1N1 23
influenza A virus) was introduced into pigs and established the current ‘classical’ swine 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
6
H1N1 lineage. Here, we characterize the functional consequences of restoring this ‘lost’ 1
function in the 2009 pandemic H1N1 virus with specific regard to host innate immune 2
suppression, virus pathogenicity and virus transmissibility in mouse and ferret animal 3
models. 4
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
7
MATERIALS AND METHODS 1
2
Cells. 293T (human), A549 (human), MDCK (canine), and PK-15 (swine) cells were 3
purchased from the American Type Culture Collection (ATCC, VA, USA) and 4
maintained in DMEM supplemented with 10% FBS, 100 units/mL of penicillin, and 100 5
µg/mL of streptomycin (GIBCO Life Technologies, CA, USA). Cryopreserved primary 6
human tracheobronchial epithelial cells (HTBEs; Clonetics, Lonza, Walkersville, MD, 7
USA) were cultivated as previously described (14). Briefly, after thawing, cells were 8
passaged twice in bronchial epithelial growth medium (BEGM; Clonetics, Lonza, 9
Walkersville, MD, USA) supplemented with retinoic acid. For full differentiation and 10
generation of an air-liquid interface, cells were seeded onto collagen-coated 12 mm 11
Transwell-Clear Permeable filters (0.4 µm pores, Corning Inc., MA, USA; collagen I 12
from human placenta, Sigma-Aldrich, MO, USA) at a density of 2 x 105 cells/filter. Cells 13
were submerged for 1 week in a 1:1 mixture of DMEM and BEGM (Gray’s medium) 14
containing necessary supplements and growth factors. At confluence, medium was 15
removed from the apical surface, and cells were maintained at the air-liquid interface for 16
at least 2 weeks. Medium was replaced every second day when cells were submerged, 17
and daily when cells were incubated at the air-liquid interface. 18
19
Plasmids. Mammalian expression constructs for untagged NS1 under control of the 20
chicken β-actin promoter [pCAGGS vector (41)] have been described previously for
21
A/Puerto Rico/8/34 [PR/34 (55)], A/Texas/36/91 [Tx/91 (28)], A/Brevig Mission/1/18 22
[BM/18 (3)], and A/Swine/Texas/4199-2/98 [Sw/Tx/98 (11)]. To generate an expression 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
8
construct for A/California/04/09 NS1 (Cal/09), cDNA encoding this NS1 protein was 1
ligated into pCAGGS using the EcoRI and NheI restriction sites. Four-primer overlap 2
PCR was used to introduce site-directed point mutations into the NS1-encoding cDNAs. 3
All NS1-encoding cDNAs also contained silent mutations in the splice
acceptor site in 4
order to prevent expression of NS2/NEP (3). A pCAGGS vector expressing GST was 5
kindly provided by Luis Martínez-Sobrido (University of Rochester Medical Center, NY, 6
USA). A pCAGGS vector expressing C-terminal FLAG-tagged human CPSF30 has been 7
described previously (28). T7-driven expression constructs for wild-type or mutant 8
Cal/09 NS1 proteins were generated by ligating the appropriate PCR-amplified cDNAs 9
between the NcoI and BamHI restriction sites of pTM1 (37). The pcDNA3 plasmid 10
encoding HA-tagged Tx/91 NS1 has been described previously (28). The reporter 11
plasmid carrying the firefly luciferase (FF-Luc) gene under control of the IFNβ promoter 12
(p125Luc) was kindly provided by Takashi Fujita (Kyoto University, Japan) (60). The 13
reporter plasmid carrying the Renilla luciferase gene (REN-Luc) under control of the 14
constitutively active HSV-TK promoter (pRL-TK) was purchased from Promega, WI, 15
USA. The identity of each construct generated or used was confirmed by sequencing. 16
17
Viruses. Stocks of Sendai virus (SeV; Cantell strain) were propagated in 10-day old 18
embryonated chicken eggs. The reference 2009 pandemic H1N1 influenza A virus 19
isolate, A/California/04/09 (Cal/09), was obtained from the Centers for Disease Control 20
and Prevention (CDC Atlanta, GA, USA), and propagated in MDCK cells. Recombinant 21
rCal/09 (wild-type, WT) was rescued according to previously reported protocols (10, 17, 22
47), with minor modifications. Briefly, 7 bi-directional pDZ-based Cal/09 RNA 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
9
expression plasmids (HA, NA, M, NP, PA, PB1, and PB2) were co-transfected with a 1
pPolI-based Cal/09 NS vRNA expression plasmid into 293T cells. At 24 h post-2
transfection, growth medium was replaced with serum-free medium containing 1 µg/mL 3
TPCK-treated trypsin (Sigma-Aldrich, MO, USA) and MDCK cells were added to the 4
culture. At 72 h post-transfection, rescue supernatant was subjected to plaque assay on 5
MDCK cells in order to obtain clonal isolates. For rescue of the rCal/09 TripleMut virus 6
(NS1 amino-acid substitutions R108K, E125D, and G189D), site-directed mutagenesis 7
was used to introduce the required mutations into pPolI-Cal/09-NS using the QuikChange 8
II Site Directed Mutagenesis Kit (Stratagene, TX, USA). The identity of the construct 9
was confirmed by DNA sequencing. Rescue of the mutant virus was performed as 10
described for wild-type. Rescued viruses were propagated in MDCK cells, and the 11
genotypes of the rCal/09 WT and TripleMut viruses were confirmed by RT-PCR and 12
sequencing of the entire NS segments. As required by the Mount Sinai School of 13
Medicine biosafety committee, work involving Cal/09 viruses was carried out in either a 14
USDA and CDC-approved BSL3+ containment laboratory, or a dedicated BSL2 15
laboratory with personnel adhering to BSL3 working practices. 16
17
Reporter assays. For analysis of IFNβ promoter activation, 293T cells in 12-well plates 18
were transfected with 25 ng of p125Luc and 2 µg of the
indicated NS1 (or GST) 19
expression plasmid using FuGENE6 (Roche, WI, USA). After 16 h, the cells were 20
infected with approximately 1 PFU/cell of SeV for 12 h. Cells were harvested and lysed 21
in 200 µL of passive lysis buffer (Promega, WI, USA), and FF-Luc activity was 22
determined using a luminometer. To measure gene expression under control of the
23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
10
constitutively active HSV-TK promoter, 293T or PK-15 cells in 12-well plates were co-1
transfected with 50 ng of pRL-TK and 2 µg of the indicated NS1 (or GST) constructs
2
using FuGENE6 (Roche, WI, USA). Expression of REN-Luc activity was measured 24 h 3
post-transfection as directed by the manufacturer (Promega, WI, USA). All transfections
4
were carried out in triplicate, and experiments were independently repeated at least twice. 5
6
SDS-PAGE, Western blot and immunoprecipitations. Cells were lysed in disruption 7
buffer (6 M urea, 2 M β-mercaptoethanol, 4% SDS), sonicated to shear nucleic acids, and 8
boiled for 5 min prior to polypeptide separation by SDS-PAGE on 4-15% Tris-HCl 9
gradient gels (Bio-Rad Laboratories, CA, USA). Proteins were detected either by 10
Coomassie blue staining or by Western blot analysis following transfer to polyvinylidene 11
difluoride (PVDF) membranes. The rabbit anti-tubulin and mouse anti-FLAG antibodies 12
were from Sigma-Aldrich, MO, USA. Rabbit polyclonal anti-serum to detect NP, and 13
rabbit polyclonal anti-serum raised against a GST-NS1(RBD) fusion protein (that detects 14
both GST and NS1) have been described previously (50). To assess the interaction of 15
different Cal/09 NS1 constructs with FLAG-tagged CPSF30,
35S-methionine labeled NS1 16
proteins were synthesized in vitro from pTM1-Cal/09-NS1 expression plasmids using a 17
TNT transcription/translation kit (Promega, WI, USA). 293T cells transiently-expressing 18
FLAG-CPSF30 were lysed in 50 mM Tris-HCl (pH 7.8), 500 mM NaCl, 5 mM EDTA, 19
and 0.5% NP-40, supplemented with a complete Mini protease inhibitor cocktail (Roche, 20
IN, USA). Following sonication, cleared cell lysates expressing FLAG-CPSF30 (or not) 21
were incubated for 2 h at 4°C with the radiolabeled NS1 proteins and 25 µL of anti-FLAG 22
affinity resin (Sigma-Aldrich, MO, USA). After extensive washing, precipitated proteins 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
11
were dissociated from the resin using disruption buffer and analyzed by SDS-PAGE 1
followed by Coomassie blue staining or Western blot. The 35
S-methionine labeled NS1 2
proteins were detected by phosphorimager analysis. 3
4
Poly(rI):poly(rC)-Sepharose co-precipitations. A549 cells were infected with the 5
appropriate virus at an MOI of 5 PFU/cell, and lysates were prepared after 16 h in 50 mM 6
Tris-HCl (pH 7.8), 500 mM NaCl, 5 mM EDTA, and 0.5% NP-40, supplemented with a 7
complete Mini protease inhibitor cocktail (Roche, IN, USA). Following sonication, 8
clarified lysates were incubated for 2 h at 4°C with poly(rI):poly(rC) (pI:C) Sepharose 9
[generated as described previously (6)] or Sepharose only. After extensive washing, 10
precipitated proteins were dissociated from the resin using disruption buffer and analyzed 11
by SDS-PAGE followed by Western blot. 12
13
Virus growth analysis. Cultures of primary differentiated HTBEs were washed 14
extensively with PBS (to remove accumulated mucus), and infected in triplicate with the 15
appropriate virus at an MOI of 0.001 PFU/cell (in the absence of exogenous TPCK-16
treated trypsin). Following infection, cells were maintained at the air-liquid interface. 17
Culture supernatants were harvested at various times post-infection by addition of PBS to 18
the apical surface of the cells for 30 min. Growth analysis in PK-15 cells was essentially 19
the same, albeit no air-liquid interface was established and cells were maintained 20
throughout in Opti-MEM (GIBCO Life Technologies, CA, USA) supplemented with 3% 21
bovine serum albumin and 1 µg/mL TPCK-treated trypsin. Virus titers were determined 22
by plaque assay on MDCK cells. Plaques were visualized either by crystal violet staining, 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
12
or by immunostaining with a rabbit polyclonal anti-serum raised against whole-1
inactivated virus (A/Puerto Rico/8/34) (50). 2
3
Quantitative real-time PCR (qPCR). Undifferentiated HTBEs in 12-well plates were 4
infected in triplicate (or mock) with virus at an MOI of 5 PFU/cell. At various times post-5
infection, cells were lysed and total RNA extracted using the RNeasy Mini Kit (Qiagen, 6
CA, USA) according to the manufacturer’s instructions. All samples were subjected to 7
DNAse treatment (Qiagen, CA, USA). For reverse transcription, 3 µg of total RNA and 8
0.5 µg oligo(dT) primer (in a total volume of 12.5 µL) was heated for 10 min at 68°C. 9
After addition of enzyme reaction buffer, 20 units RiboLock RNase inhibitor, 20 nmol 10
dNTPs, and 200 units RevertAid M-MuLV reverse transcriptase (Fermentas Inc., MD, 11
USA), samples were heated at 42°C for 1 h. Reverse transcriptase activity was 12
subsequently inactivated at 70°C for 10 min. The resulting cDNA product acted as the 13
template for qPCR using the Brilliant QPCR SYBR Green Mastermix system (Stratagene, 14
TX. USA) and a Roche Light Cycler 480. The assay was performed according to the 15
manufacturers’ instructions, and the fragment of interest was amplified in 40 cycles. 16
Primer sequences used are available upon request. Samples were tested in triplicate, and 17
cDNA levels normalized to 18S RNA levels. Fold-induction over the mock-infected 18
samples was calculated according to the 2-∆∆CT
-method (32). 19
20
Mouse experiments. All mouse procedures were performed in accordance with the 21
Institutional Animal Care and Use Committee guidelines of Mount Sinai School of 22
Medicine, and were carried out in a BSL3 containment laboratory. 7-week old female 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
13
congenic B6.A2G-Mx1 mice (53) carrying intact Mx1 alleles on the C57BL/6 background 1
(kindly provided by Peter Staeheli, University of Freiburg, Germany and bred locally) or 2
DBA/2J mice (The Jackson Laboratory, ME USA) were anaesthetized using ketamine-3
xylazine and intranasally infected with the indicated dose of virus diluted in 50 µL of 4
PBS. Body weights were subsequently measured daily for 2 weeks post-infection. Mice 5
showing more than 25% weight loss were considered to have reached the experimental 6
endpoint and were humanely euthanized. Lungs were excised from euthanized mice and 7
homogenized in 1 ml PBS supplemented with 0.2% bovine serum albumin and 8
penicillin/streptomycin using a mechanical homogenizer (MP Biochemicals, OH, USA). 9
After centrifugation (10,000 x g, 5 min, 4°C), resulting supernatants were used to 10
determine viral titer, and pelleted debris was used for RNA extraction using TRIzol 11
(Invitrogen, CA, USA). 12
13
Ferret experiments. All ferret procedures were performed in accordance with the 14
Institutional Animal Care and Use Committee guidelines of the University of Maryland, 15
and were carried out in a USDA-approved BSL3+ containment laboratory. The basic 16
transmission study scheme consisted of duplicate groups of three 6-8 month old female 17
Fitch ferrets (Triple F Farms, PA USA): one infected (intranasal with 106 TCID50 of virus 18
in PBS, 600 µL per nostril), one direct contact and one respiratory droplet contact. Nasal 19
washes were collected daily. For tissue collection, infected ferrets were humanely 20
euthanized on day 5 post-infection, and nasal turbinate, trachea and lung samples were 21
collected. All experimental details were essentially as described previously (51), and 22
virus samples from nasal washes and tissues were titrated on MDCK cells. 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
14
RESULTS 1
2
IFN-antagonistic properties of the 2009 H1N1 virus NS1 protein. We tested the 3
ability of the swine-origin 2009 pandemic H1N1 virus NS1 protein to limit production of 4
IFNβ in human cells, and compared it with a panel of other NS1 proteins known to 5
inhibit (or not (22, 28)) general gene expression by binding CPSF30. 293T cells were 6
transfected with a firefly-luciferase IFNβ promoter reporter construct together with 7
expression plasmids for the NS1 proteins of A/Puerto Rico/8/34 [PR/34], A/Texas/36/91 8
[Tx/91], A/Brevig Mission/1/1918 [BM/18], A/Swine/Texas/4199-2/98 [Sw/Tx/98], or a 9
representative isolate of the 2009 pandemic H1N1 influenza A virus, A/California/04/09 10
[Cal/09]. A GST expression plasmid served as a negative control. Sixteen hours post-11
transfection, cells were infected with SeV for a further 12 h prior to analysis of FF-12
luciferase activity. As shown in FIG. 1, SeV infection induced robust amounts of IFNβ 13
promoter-driven FF-luciferase activity in GST-expressing cells (set to 100%), but not in 14
cells expressing any of the NS1 proteins (< 4%), including the NS1 protein of the 2009 15
pandemic H1N1 influenza A virus (Cal/09). Despite this clear inhibition by all NS1 16
proteins tested, there was a minor (yet statistically significant) difference: whilst both the 17
NS1 proteins of Tx/91 and BM/18 completely blocked induction of FF-luciferase activity, 18
the NS1 proteins of PR/34, Sw/Tx/98, and Cal/09 were unable to inhibit a small fraction 19
of activity (i.e. 3-4%; FIG. 1 inset). Given that a major phenotypic difference between 20
the NS1 proteins of PR/34 and Tx/91 is their ability to bind CPSF30 and thus to limit 21
gene expression (28), we postulated that the incomplete block demonstrated by both 22
Sw/Tx/98 and Cal/09 NS1 proteins was, like PR/34, due to an inability to inhibit 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
15
CPSF30. We suspect that pre-transcriptional block of TRIM25/RIG-I by NS1 (11, 15, 28, 1
36, 44, 45, 55) accounts for the majority of IFNβ-promoter inhibition in our assay. 2
3
The 2009 H1N1 virus NS1 protein is unable to block general host gene expression in 4
either human or swine cells. To formally test whether the Cal/09 NS1 protein is unable 5
to block general host gene expression, we co-transfected a constitutively-active Renilla-6
luciferase reporter construct (pRL-TK) with each of the indicated NS1 constructs and 7
measured total Renilla-luciferase activity 24 h later. Previous data from our laboratory 8
have demonstrated a clear link between the ability of NS1 proteins to bind CPSF30 and 9
their ability to block Renilla-luciferase reporter activity in this assay (28). As shown in 10
FIG. 2A, only the NS1 proteins of Tx/91 and BM/18 efficiently inhibited Renilla-11
luciferase activity in human 293T cells, whilst the NS1 proteins of PR/34 and the swine-12
derived Sw/Tx/98 and Cal/09 (like GST) were largely deficient in this function. We also 13
analyzed the total amounts of GST or NS1 protein in these transfected cell lysates. As 14
expected, the NS1 proteins that efficiently blocked Renilla-luciferase activity (Tx/91 and 15
BM/18) were barely detectable (FIG. 2B), presumably because the small amount of NS1 16
protein made is sufficient to limit maturation of further RNA polymerase II transcripts 17
from its own transfected plasmid, as well as from the Renilla-luciferase plasmid. In 18
contrast, the NS1 proteins of PR/34, Sw/Tx/98, and Cal/09 expressed well (FIG. 2B), 19
probably as they are unable to block their own expression. These observations are entirely 20
consistent with previous results obtained comparing NS1 proteins able (or not) to bind 21
and inhibit CPSF30 (28). 22
23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
16
Given that both Sw/Tx/98 and Cal/09 NS1 proteins are derived from swine-origin 1
viruses, and therefore may specifically not function in human cells, we tested whether 2
these NS1 proteins were better adapted to block general gene expression in swine cells. 3
Thus, we also performed the Renilla-luciferase reporter activity assays in transfected PK-4
15 cells, a porcine kidney epithelial cell-line. However, as shown in FIGs. 2C and 2D, 5
the results obtained were essentially identical to those obtained using a human cell-line: 6
Tx/91 and BM/18 NS1 proteins inhibited gene expression (both of the Renilla-luciferase 7
reporter and themselves), whilst the NS1 proteins of PR/34, Sw/Tx/98, and Cal/09 did 8
not. This suggests that the inability of swine-origin influenza virus NS1 proteins to block 9
gene expression is not restricted to non-swine cells. Furthermore, in agreement with the 10
hypothesis that failure of these NS1 proteins to block gene expression is related to their 11
inability to bind CPSF30, the CPSF30 amino-acid sequences between human and swine 12
are 99.6% identical (243/244 residues, G. Pisanelli and A. G-S, unpublished). 13
14
Substitution of residues 108, 125, and 189 in the 2009 H1N1 virus NS1 protein 15
restore its ability to inhibit general gene expression and to bind CPSF30. The 16
interaction between NS1 and CPSF30 is primarily mediated by the NS1 effector domain 17
(ED, residues 85-203) and two zinc-fingers of CPSF30 (the F2F3 region) (8, 57). Based 18
on the assumption that the NS1 phenotypes were due to their differential abilities to bind 19
CPSF30, we aligned the amino-acid sequences of the NS1-EDs used in this study (FIG. 20
3A). Overall, the EDs are highly conserved, and the few amino-acid residues of NS1 that 21
have been experimentally implicated in CPSF30 binding to date (e.g. 103, 106, and 184-22
188 (8, 28, 31, 42, 56), FIG. 3A, gray shading) are no different between the NS1 23
proteins of BM/18 or Tx/91 (which block gene expression) and Sw/Tx/98 or Cal/09 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
17
(which lack this function). Furthermore, residues 103 and 106 (which determine lack of 1
CPSF30 binding for PR/34 NS1) are also human ‘binding’ consensus for the Cal/09 NS1 2
protein. In fact, there are only nine amino-acid positions in the ED that consistently differ 3
between the swine-origin viruses and all the ‘human-like’ viruses (FIG. 3A, black 4
boxes). Notably, three of these residues are located at the direct interface between NS1 5
and CPSF30 (8): 108, 125, and 189 (FIGs. 3B and 3C), although none of these NS1 6
residues have previously been experimentally shown to contribute towards CPSF30 7
binding. 8
9
We constructed Cal/09 NS1 expression plasmids with each of the following single amino-10
acid substitutions: R108K, E125D, and G189D (Cal/09 residues were changed to human 11
influenza A virus consensus), and tested their ability to block general gene expression in 12
our Renilla-luciferase reporter activity assay. In human 293T cells, each mutant Cal/09 13
NS1 protein was able to limit luciferase activity significantly better than the wild-type 14
(WT) Cal/09 NS1, with the G189D mutant inhibiting approximately 50% activity (FIG. 15
4A). However, as compared with Tx/91 NS1, the block was still relatively poor. We 16
therefore made combinations of the mutations and tested their effect on gene expression. 17
Although luciferase inhibition with the dual R108K/G189D mutant was better than WT, 18
only the Cal/09 NS1 with all three amino-acid substitutions (R108K, E125D, and G189D; 19
TripleMut) completely blocked gene expression to a level comparable to Tx/91 NS1 20
(FIG. 4A). As confirmation, we also observed that NS1 proteins best able to block 21
luciferase activity also limited their own expression (FIG. 4B). This effect was not 22
limited to human cells, as the same overall result was obtained in swine PK-15 cells 23
(FIGs. 4C and 4D, respectively). 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
18
1
In order to assess whether the three R108K, E125D, and G189D mutations also increased 2
binding of Cal/09 NS1 to CPSF30, we tested the co-precipitation of in vitro transcribed 3
and translated 35
S-methionine labeled NS1 (WT or TripleMut) with transiently expressed 4
FLAG-tagged CPSF30. Using high stringency conditions (500 mM NaCl), no detectable 5
WT Cal/09 NS1 protein could be co-precipitated with FLAG-CPSF30 (FIG. 5A). 6
However, the TripleMut Cal/09 NS1 clearly bound to FLAG-CPSF30 (FIG. 5A). The 7
interaction of TripleMut Cal/09 NS1 with FLAG-CPSF30 was comparable to that of 8
Tx/91 NS1 (FIG. 5A). We also performed immunoprecipitations using lower salt 9
concentrations (200 mM NaCl), under which conditions a small fraction of WT Cal/09 10
NS1 was found to co-precipitate with FLAG-CPSF30, although binding efficiency was 11
clearly enhanced for the TripleMut NS1 (FIG. 5B). The three mutations also appeared to 12
have minimal overall effect on the ability of Cal/09 NS1 to bind synthetic dsRNA, as 13
revealed by pI:C pull-down assays (FIG. 5C). Together with data from the Renilla-14
luciferase reporter activity assays, our results indicate that mutation of residues 108, 125, 15
and 189 in Cal/09 NS1 to human consensus restores the ability of this NS1 protein to bind 16
more efficiently to CPSF30 and consequently to inhibit cellular gene expression. Thus, 17
although the amino-acid substitutions made are conservative, they clearly have a dramatic 18
impact on a specific NS1 function. It is likely that side-chain length, as well as charge, 19
determine overall NS1:CPSF30 complex stability. 20
21
In vitro characterization of a recombinant 2009 H1N1 virus expressing NS1 with the 22
R108K, E125D, and G189D amino-acid substitutions. We cloned and established a 23
complete reverse genetics system for the Cal/09 influenza virus isolate (16). The rescued 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
19
recombinant WT Cal/09 virus (rCal/09 WT) grew with identical kinetics to the parental 1
non-recombinant Cal/09 isolate in primary differentiated HTBEs (FIG. 6A). We also 2
used this reverse genetics system to generate a recombinant mutant Cal/09 virus 3
expressing NS1 with the R108K, E125D, and G189D amino-acid substitutions 4
(TripleMut). Unavoidably, by generating the G189D amino-acid change in NS1, a V32I 5
amino-acid substitution also occurs in NEP/NS2. However, this very conservative change 6
reverts the NEP/NS2 residue back to human virus consensus, and would occur even if 7
G189D arose naturally. It is unknown what effect such an amino-acid substitution has on 8
NEP/NS2, as this residue lies outwith any previously characterized functional domain. In 9
vitro characterization of the engineered rCal/09 TripleMut virus revealed that both this 10
and rCal/09 WT virus had similar plaque phenotypes on MDCK cells (FIG. 6B). 11
Furthermore, Western blot analysis confirmed that viral protein expression in infected 12
MDCKs was identical for both viruses (FIG. 6C). We also assessed replication of the 13
rescued viruses in primary differentiated HTBEs and the swine PK-15 cell-line. 14
Surprisingly, and in contrast to our initial hypothesis, the three NS1 mutations did not 15
confer a selective growth advantage to rCal/09 TripMut over rCal/09 WT in the primary 16
human cells (FIG. 6D), nor was this mutant significantly attenuated in the swine cell-line 17
(FIG. 6E). These data also indicate that the single conservative amino-acid substitution 18
(V32I) in NS2/NEP does not affect the replication of Cal/09 in vitro. 19
20
We next tested the ability of the rCal/09 TripleMut virus to limit expression of IFNβ and 21
IFN-stimulated genes during infection. Primary undifferentiated HTBEs were infected at 22
an MOI of 5 PFU/cell with rCal/09 WT or rCal/09 TripleMut. At various times post-23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
20
infection, total RNA was extracted and relative fold-induction of the indicated viral and 1
cellular mRNAs was quantified by qPCR. As shown in FIG. 7A, the infectious titers 2
reached after 24 h by both viruses were identical. In addition, the levels of viral NA 3
mRNA increased at the same rate for both rCal/09 WT and the rCal/09 TripleMut viruses 4
(FIG. 7B), consistent with the equal growth kinetics of these viruses observed in both 5
primary differentiated HTBE cells and the swine PK-15 cell-line (FIG. 6). Nevertheless, 6
it should be noted that both replication and cytokine responses can differ depending upon 7
the differentiation state of primary cells (7). However, as expected, the amount of IFNβ 8
mRNA produced during infection with the rCal/09 TripleMut virus was significantly 9
lower than that produced by the rCal/09 WT virus (FIG. 7C). Furthermore, the mRNA 10
levels of IFN-stimulated genes such as OAS, MxA, and IP10 (FIGs. 7D-F, respectively) 11
were also significantly lower in rCal/09 TripleMut virus-infected cells, probably as a 12
result of both their direct inhibition (28) and the indirect effect of IFNβ limitation. We 13
attribute this general immune-antagonistic phenotype to the enhanced capability of the 14
rCal/09 TripleMut NS1 protein to bind CPSF30 and block cellular gene expression. Such 15
data are in full agreement with our observations using transiently-expressed WT and 16
TripleMut Cal/09 NS1 proteins in the absence of virus infection (FIG. 4). Furthermore, 17
these results indicate that for WT Cal/09 virus, the viral polymerase is unable to 18
compensate wholly for the WT NS1 deficiency (e.g. by the viral cap-snatching activity 19
(27, 46), or virus-induced RNA polymerase II degradation (48, 58)). Nevertheless, as 20
reported for other influenza A viruses with NS1 polymorphisms that also preclude direct 21
CPSF30 binding (29, 56), it may be that the cognate WT Cal/09 viral polymerase 22
complex allows a minor fraction of WT NS1 to associate with CPSF30 during infection, 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
21
thereby contributing to partial innate immune suppression at the level of pre-mRNA 1
processing. Any such inhibition is likely to be in addition to a pre-transcriptional block by 2
NS1 at the level of RIG-I/TRIM25 (11). 3
4
In vivo characterization of a recombinant 2009 H1N1 virus expressing NS1 with the 5
R108K, E125D, and G189D amino-acid substitutions. To test whether increased ability 6
of NS1 to block general gene expression would affect in vivo viral replication and/or 7
pathogenicity, we compared the rCal/09 WT and rCal/09 TripleMut viruses in a mouse 8
model. Initial studies using 5x106 PFU of rCal/09 WT or rCal/09 TripleMut showed that 9
both these viruses replicated to similar average titers in the lungs of infected congenic 10
B6.A2G-Mx1 mice [Day 2: 5.5x105 PFU/mL (WT) vs. 3.5x10
5 PFU/mL (TripleMut); 11
Day 4: 4.2x103 PFU/mL (WT) vs. 2.0x10
3 PFU/mL (TripleMut)]. However, no 12
significant body weight loss after infection was observed at this dose in these mice (data 13
not shown). DBA/2J mice were therefore selected for subsequent experiments as they are 14
reported to be highly susceptible to influenza virus infection (4, 17, 52). Mice were 15
infected with 105 PFU of each virus (or PBS alone) and body weights were monitored 16
daily for 14 days. Surprisingly, the rCal/09 TripleMut virus caused less weight loss in 17
infected DBA2/J mice than the rCal/09 WT virus (FIG. 8A). Furthermore, although in 18
the lungs of mice infected with 104 PFU of virus the rCal/09 TripleMut appeared to reach 19
similar mean peak infectious titers to WT [FIG. 8B, day 2: 2x106 PFU/mL (TripleMut) 20
vs. 4x106 PFU/mL (WT)], the rCal/09 TripleMut virus seemed to be cleared slightly 21
faster [FIG. 8B, day 7: 2.5x103 PFU/mL (TripleMut) vs. 3x10
4 PFU/mL (WT)]. 22
Consistent with our data in infected undifferentiated HTBEs, quantitative analysis of 23
IFNβ mRNA in infected mouse lungs indicated that the rCal/09 TripleMut virus induced 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
22
less of an innate immune response than WT (FIG. 8C). However, we cannot exclude the 1
possibility that this is due to slight attenuation of the TripleMut virus in vivo rather than 2
efficient antagonism of the murine IFN system. 3
4
We also studied the replication abilities of the rCal/09 WT and rCal/09 TripleMut viruses 5
in a ferret model. Ferrets were infected with 106 TCID50 of each virus and the amount of 6
infectious virus present in different parts of the respiratory tract was determined at 5 days 7
post-infection. Consistent with the mouse data indicating that the rCal/09 TripleMut virus 8
is cleared faster than WT, less rCal/09 TripleMut virus was detected in the nasal turbinate 9
of ferrets at this time-point, as compared with WT (FIG. 9A). In addition, the amount of 10
rCal/09 TripleMut virus in the lung appeared lower than WT (FIG. 9A). However, 11
similar titers for both viruses were observed in the trachea (FIG. 9A). As previously 12
described (51), the ferret model also enabled us to assess both the direct contact and 13
aerosol respiratory droplet transmission dynamics of the two viruses. By analyzing daily 14
nasal washes of infected and contact ferrets, we observed that total rCal/09 WT virus 15
shedding was slightly higher than that of rCal/09 TripleMut in infected ferrets (FIGs. 9B 16
and 9C), possibly due to faster clearance of the TripleMut virus as seen with the DBA/2J 17
mice. Nevertheless, the rCal/09 TripleMut virus still transmitted as efficiently as WT to 18
other ferrets by both direct contact and respiratory droplet routes (FIGs. 9B and 9C). 19
These data suggest that, in contrast to our observations in vitro, gain-of-function 20
mutations in NS1 that enhance its ability to block general gene expression may reduce the 21
overall replication and pathogenicity of Cal/09 in vivo. 22
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
23
DISCUSSION 1
2
Whilst characterizing the 2009 pandemic H1N1 influenza A virus, we found that the NS1 3
protein of this virus lacks the ability to block general host gene expression in both human 4
and swine cells. This is due to its inefficient binding to cellular CPSF30, a property 5
otherwise maintained in circulating human influenza A viruses (56) that is reported to 6
contribute towards suppression of host innate immune responses (8, 42). By identifying 7
and making novel human-adaptive mutations in NS1, we were able to introduce this 8
function into a recombinant prototype 2009 H1N1 virus isolate, Cal/09. As with other 9
influenza A viruses shown naturally to be defective in blocking CPSF30 function [e.g. 10
A/Hong Kong/483/97 [H5N1] (56)], we found that restoration of CPSF30 binding 11
resulted in a virus that induces less IFNβ mRNA (as well as other innate immune 12
mRNAs) during infection. 13
14
In order to restore efficient CPSF30 binding, we rationally mutated three residues of 15
Cal/09 NS1 to human H1N1 virus consensus based on the recently published crystal 16
structure of an NS1-ED in complex with CPSF30-F2F3 (8). However, other 17
uncharacterized amino-acid changes may arise naturally that also contribute to restoration 18
of CPSF30 binding independently of the three presented here. Nevertheless, these three 19
amino-acids (R108, E125, and G189) are highly conserved in the NS1 proteins of 20
‘classically-derived’ swine influenza A viruses, particularly within the H1 subtype 21
(TABLE 1). The viruses isolated from swine that have other amino-acids at these 22
positions are either ‘spill-over’ viruses from other species (only a small percentage of 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
24
human and avian H1 influenza A viruses have the swine consensus), or swine viruses of 1
the ‘European avian-like’ lineage. Based on this conservation, and the poor ability of 2
Sw/Tx/98 NS1 to block general gene expression, we hypothesize that lack of binding and 3
inhibition of CPSF30 is a general feature of ‘classically-derived’ swine influenza A virus 4
NS1 proteins. Indeed, the NS1 protein of the first influenza virus isolated from pigs 5
(A/Swine/Iowa/15/1930) already had R108, E125, and G189. This is in stark contrast to 6
its closest human relative, the 1918 pandemic influenza virus (e.g. A/Brevig 7
Mission/1/18), which had K108, D125, and D189, and clearly binds/inhibits CPSF30 8
[this study, (28, 56)]. Thus, it may be that non-binding amino-acid substitutions were 9
selected for in the pig population after introduction of the precursor virus, and have been 10
maintained in the ‘classical’ swine NS lineage until the present day. It should be noted 11
that such non-binding substitutions have yet to be detected in the ‘European avian-like’ 12
lineage of swine influenza A viruses that was established in 1979. It is possible that 13
complete swine adaptation has yet to occur for this new lineage, or that the ‘classical’ and 14
‘European avian-like’ lineages are evolving independently (9). In this latter regard, it may 15
be that lineage-specific functional adaptation in NS1 is generated by a different set of 16
mutations. We are planning to test the ability of ‘European avian-like’ NS1 proteins to 17
bind/inhibit CPSF30. 18
19
We had hypothesized that enhanced ability to limit IFNβ production would increase 20
replication of the rCal/09 virus in human cells. However, we were unable to detect an 21
increase in viral replication due to this new function, either in primary human 22
tracheobronchial epithelial cells or in a swine cell-line. Furthermore, one might have 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
25
expected the gain-of-function virus to grow better only in human cells, and worse in 1
swine cells, particularly if ability of NS1 to bind CPSF30 is a human-adaptive phenotype 2
that is selected against in pigs. Thus, it may be that our experimental set-up is not 3
sensitive enough for detecting small phenotypic differences. It is possible that studies 4
using additional primary substrates or cell-types (particularly of swine origin) could aid in 5
further dissecting the effect of these NS1 adaptations. We also cannot exclude the notion 6
that the polymorphism changes we made to the Cal/09 NS1 protein negatively affect 7
another of the multiple NS1 functions independently of their clear positive influence on 8
CPSF30 binding. For example, the alterations we made are at one of the proposed dimeric 9
interfaces of the NS1 effector domain (18), which is concomitant with the CPSF30 10
binding interface (8). Thus, slight alterations in the temporal dimeric packing of the 11
TripleMut effector domain (or even the single amino-acid change in NEP) may have 12
slightly reduced virus replication thereby countering any positive effect seen by restoring 13
NS1:CPSF30 binding. Intriguingly, given that we only made naturally-occurring 14
polymorphism changes in the NS segment, such a possibility suggests that the potential 15
“unknown function” lost by these mutations may actually be important and selected for in 16
the ‘classical’ swine lineage. 17
18
The contribution of NS1 to the higher replication efficiency and pathogenicity of the 19
2009 H1N1 virus [as compared with seasonal H1N1 viruses (24, 34, 38)] is unknown. 20
Nevertheless, the NS1 protein of this pandemic virus lacks previously postulated 21
virulence markers, such as deletion of residues 80-84 (33), a glutamic acid at position 92 22
(49), or the presence of a consensus C-terminal PDZ-domain ligand motif (25, 43). 23
Furthermore, experimental introduction of naturally-isolated specific functional mutations 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
26
in the NS1 C-terminal tail did not alter replication, pathogenicity or transmission of the 1
2009 H1N1 virus (20). In our in vivo DBA/2J mouse model of infection the gain-of-2
function (TripleMut) virus was slightly less pathogenic, and appeared to be cleared faster, 3
than the wild-type virus (which lacks the ability to inhibit CPSF30). Prima facie, these 4
data suggest that inefficient CPSF30 inhibition may actually contribute to virulence of 5
this swine-origin virus in mice. Although it has been noted that the wild-type Cal/09 virus 6
induces high amounts of certain pro-inflammatory cytokines during in vivo infections of 7
mice and cynomolgus macaques (24), it is not known if this is due solely to a defect in 8
NS1 function. In addition, it is unclear whether this exuberant immune response plays 9
any significant role in disease progression. To define lack of CPSF30 binding as a bona 10
fide ‘virulence factor’ for the 2009 pandemic H1N1 influenza A virus, we would have to 11
remove this function from an unrelated influenza A virus strain that naturally inhibits 12
CPSF30 and assess its resulting pathogenicity. It may also be that correlation of poor 13
CPSF30 binding with virulence is a mouse-specific phenotype, particularly as many 14
experimentally-derived mouse-adapted viruses have mutations that at least partially 15
destabilize the interaction of NS1 with CPSF30 (5, 28). If this is the case, it is interesting 16
that the TripleMut virus also demonstrated lower viral titers in the nasal turbinates of 17
ferrets. Of note, the mouse-adapted influenza virus strain A/WSN/33 (WSN/33) contains 18
asparagine at position 189, rather than the aspartic acid consensus for efficient CPSF30 19
binding. Thus, although WSN/33 NS1 has been shown to interact with the F2F3 domain 20
of CPSF30 (56), this may be less efficient than NS1 proteins from non-mouse adapted 21
seasonal human viruses. If enhanced virulence in mice correlates with poor CPSF30 22
binding, then this may explain a previous observation whereby introduction of the BM/18 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
27
NS1 (which binds/inhibits CPSF30 very efficiently) into a recombinant WSN/33 virus 1
significantly reduced pathogenicity in a mouse model (3). 2
3
The biological reasons for different influenza A viruses maintaining or not their ability to 4
limit general gene expression via NS1 are unclear. Nevertheless, a pattern is now 5
emerging that this may be a viral strain- and host species- specific phenotype. Our data 6
based on residues 108, 125, and 189 would strongly suggest that swine influenza viruses 7
commonly lack this function. Furthermore, analysis of residues 103 and 106 [two major 8
determinants of CPSF30 binding efficiency (28, 56)] shows that stable CPSF30 binding is 9
also generally lost in avian H6 and H9 viruses (TABLE 1), particularly in H6 and H9 10
viruses that infect quail, pheasants, and partridges, rather than ducks and chickens. This 11
observation may have already been confirmed experimentally when adapting a duck 12
influenza virus to quail, as the resulting virus had the M106T amino-acid change (23). 13
We note that a surprising proportion of 2009 H1N1 viruses (approximately 2%) isolated 14
since April 2009 also have amino-acid substitutions at positions 103 or 106 in NS1 15
(TABLE 1). This is likely due to minimal selection pressure to maintain these CPSF30 16
binding residues given that the protein lacks this function already. Remarkably, most 17
human H3 influenza A viruses appear to have the potential ‘affinity-lowering’ glutamic 18
acid residue at position 125, although presumably these NS1 proteins can all bind 19
CPSF30 to some extent. Another notable subset of NS1 proteins are those classified as 20
‘allele B’, which are exclusively found in avian influenza A viruses [reviewed in (19)]. 21
Almost all ‘allele B’ NS1 proteins have tyrosine at position 103 and arginine at position 22
108. Thus, there may be ‘sliding-scale’ of CPSF30 binding affinities from strong to weak, 23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
28
with each particular virus (in a particular host) replicating optimally with only a certain 1
level of CPSF30 inhibition. 2
3
It is still unclear whether efficient CPSF30 binding by NS1 contributes to the human 4
adaptation of influenza A viruses. However, now that the swine-origin 2009 pandemic 5
H1N1 virus is circulating in humans, we may start to see human virus consensus emerge 6
at residues 108, 125, and 189. Indeed, nine 2009 pandemic H1N1 viruses with individual 7
mutations at these positions (each with the potential to slightly increase CPSF30 binding) 8
have already been isolated, although whether they are more fit is unknown. From a public 9
health point of view, our in vivo data would suggest that in the current 2009 H1N1 viral 10
genetic background, such amino-acid changes in NS1 alone (or others that independently 11
restore even a partial phenotype) will not dramatically increase virus pathogenicity, nor 12
significantly affect virus transmissibility. Nevertheless, given our data in primary 13
undifferentiated human tracheobronchial epithelial cells, where the rCal09 TripleMut 14
virus seems better than WT at suppressing the host innate immune response, we cannot 15
exclude the possibility that potential future gain of this NS1 function will enhance 16
replication of the 2009 pandemic H1N1 virus in humans. It may therefore be particularly 17
prudent to survey newly emerging NS segment mutations, determine the corresponding 18
NS1 protein phenotype, and assess any associations with clinical outcome. 19
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
29
ACKNOWLEDGEMENTS 1
2
We are grateful to Peter Palese (Mount Sinai School of Medicine, NY, USA) for helpful 3
discussions and for critical reading of the manuscript. We thank Osman Lizardo for 4
excellent technical assistance, and the Centers for Disease Control and Prevention (CDC 5
Atlanta, GA, USA), Yoshihiro Kawaoka (University of Wisconsin-Madison, WI, USA), 6
Estanislao Nistal-Villán (Mount Sinai School of Medicine, NY, USA), Randy Albrecht 7
(Mount Sinai School of Medicine, NY, USA), Takashi Fujita (Kyoto Univeristy, Japan), 8
Peter Staeheli (University of Freiburg, Germany), and Luis Martínez-Sobrido (University 9
of Rochester Medical Center, NY, USA) for some of the reagents used in this study. This 10
work was partially supported by CRIP, an NIAID funded Center for Research in 11
Influenza Pathogenesis (contract number HHSN266200700010C), and by NIAID grants 12
RO1AI46954, U19AI83025 and PO1AI58113 (to AG-S). JS is supported by a career 13
development fellowship from the Northeast Biodefense Center (U54 AI057158). ACL is 14
a Parker B. Francis Fellow in pulmonary research. 15
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
30
REFERENCES 1
2
1. 2009. Swine influenza A (H1N1) infection in two children--Southern California, 3
March-April 2009. MMWR Morb Mortal Wkly Rep 58:400-2. 4
2. Babiuk, S., R. Albrecht, Y. Berhane, P. Marszal, J. A. Richt, A. Garcia-5
Sastre, J. Pasick, and H. Weingartl. 2009. 1918 and 2009 H1N1 influenza are 6
not pathogenic in birds. J Gen Virol. 7
3. Basler, C. F., A. H. Reid, J. K. Dybing, T. A. Janczewski, T. G. Fanning, H. 8
Zheng, M. Salvatore, M. L. Perdue, D. E. Swayne, A. Garcia-Sastre, P. 9 Palese, and J. K. Taubenberger. 2001. Sequence of the 1918 pandemic 10
influenza virus nonstructural gene (NS) segment and characterization of 11
recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 12
98:2746-51. 13
4. Boon, A. C., J. deBeauchamp, A. Hollmann, J. Luke, M. Kotb, S. Rowe, D. 14
Finkelstein, G. Neale, L. Lu, R. W. Williams, and R. J. Webby. 2009. Host 15
genetic variation affects resistance to infection with a highly pathogenic H5N1 16
influenza A virus in mice. J Virol 83:10417-26. 17
5. Brown, E. G., H. Liu, L. C. Kit, S. Baird, and M. Nesrallah. 2001. Pattern of 18
mutation in the genome of influenza A virus on adaptation to increased virulence 19
in the mouse lung: identification of functional themes. Proc Natl Acad Sci U S A 20
98:6883-8. 21
6. Cardenas, W. B., Y. M. Loo, M. Gale, Jr., A. L. Hartman, C. R. Kimberlin, 22
L. Martinez-Sobrido, E. O. Saphire, and C. F. Basler. 2006. Ebola virus VP35 23
protein binds double-stranded RNA and inhibits alpha/beta interferon production 24
induced by RIG-I signaling. J Virol 80:5168-78. 25
7. Chan, R. W., K. M. Yuen, W. C. Yu, C. C. Ho, J. M. Nicholls, J. S. Peiris, and 26
M. C. Chan. 2010. Influenza H5N1 and H1N1 virus replication and innate 27
immune responses in bronchial epithelial cells are influenced by the state of 28
differentiation. PLoS One 5:e8713. 29
8. Das, K., L. C. Ma, R. Xiao, B. Radvansky, J. Aramini, L. Zhao, J. Marklund, 30
R. L. Kuo, K. Y. Twu, E. Arnold, R. M. Krug, and G. T. Montelione. 2008. 31
Structural basis for suppression of a host antiviral response by influenza A virus. 32
Proc Natl Acad Sci U S A 105:13093-8. 33
9. Dunham, E. J., V. G. Dugan, E. K. Kaser, S. E. Perkins, I. H. Brown, E. C. 34
Holmes, and J. K. Taubenberger. 2009. Different evolutionary trajectories of 35
European avian-like and classical swine H1N1 influenza A viruses. J Virol 36
83:5485-94. 37
10. Fodor, E., L. Devenish, O. G. Engelhardt, P. Palese, G. G. Brownlee, and A. 38
Garcia-Sastre. 1999. Rescue of influenza A virus from recombinant DNA. J 39
Virol 73:9679-82. 40
11. Gack, M. U., R. A. Albrecht, T. Urano, K. S. Inn, I. C. Huang, E. Carnero, 41
M. Farzan, S. Inoue, J. U. Jung, and A. Garcia-Sastre. 2009. Influenza A virus 42
NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral 43
RNA sensor RIG-I. Cell Host Microbe 5:439-49. 44
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
31
12. Garcia-Sastre, A., A. Egorov, D. Matassov, S. Brandt, D. E. Levy, J. E. 1
Durbin, P. Palese, and T. Muster. 1998. Influenza A virus lacking the NS1 gene 2
replicates in interferon-deficient systems. Virology 252:324-30. 3
13. Garten, R. J., C. T. Davis, C. A. Russell, B. Shu, S. Lindstrom, A. Balish, W. 4
M. Sessions, X. Xu, E. Skepner, V. Deyde, M. Okomo-Adhiambo, L. 5
Gubareva, J. Barnes, C. B. Smith, S. L. Emery, M. J. Hillman, P. Rivailler, J. 6
Smagala, M. de Graaf, D. F. Burke, R. A. Fouchier, C. Pappas, C. M. 7
Alpuche-Aranda, H. Lopez-Gatell, H. Olivera, I. Lopez, C. A. Myers, D. Faix, 8
P. J. Blair, C. Yu, K. M. Keene, P. D. Dotson, Jr., D. Boxrud, A. R. Sambol, 9
S. H. Abid, K. St George, T. Bannerman, A. L. Moore, D. J. Stringer, P. 10
Blevins, G. J. Demmler-Harrison, M. Ginsberg, P. Kriner, S. Waterman, S. 11
Smole, H. F. Guevara, E. A. Belongia, P. A. Clark, S. T. Beatrice, R. Donis, J. 12
Katz, L. Finelli, C. B. Bridges, M. Shaw, D. B. Jernigan, T. M. Uyeki, D. J. 13 Smith, A. I. Klimov, and N. J. Cox. 2009. Antigenic and genetic characteristics 14
of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 15
325:197-201. 16
14. Gray, T. E., K. Guzman, C. W. Davis, L. H. Abdullah, and P. Nettesheim. 17
1996. Mucociliary differentiation of serially passaged normal human 18
tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 14:104-12. 19
15. Guo, Z., L. M. Chen, H. Zeng, J. A. Gomez, J. Plowden, T. Fujita, J. M. Katz, 20
R. O. Donis, and S. Sambhara. 2007. NS1 protein of influenza A virus inhibits 21
the function of intracytoplasmic pathogen sensor, RIG-I. Am J Respir Cell Mol 22
Biol 36:263-9. 23
16. Hai, R., M. Schmolke, Z. T. Varga, B. Manicassamy, T. T. Wang, J. A. 24
Belser, M. B. Pearce, A. Garcia-Sastre, T. M. Tumpey, and P. Palese. 2010. 25
PB1-F2 expression by the 2009 pandemic H1N1 influenza virus has minimal 26
impact on virulence in animal models. J Virol. 27
17. Hai, R., M. Schmolke, Z. T. Varga, B. Manicassamy, T. T. Wang, J. A. 28
Belser, M. B. Pearce, A. Garcia-Sastre, T. M. Tumpey, and P. Palese. in press. 29
PB1-F2 expression by the 2009 pandemic H1N1 influenza virus has minimal 30
impact on virulence in animal models. J Virol. 31
18. Hale, B. G., W. S. Barclay, R. E. Randall, and R. J. Russell. 2008. Structure of 32
an avian influenza A virus NS1 protein effector domain. Virology 378:1-5. 33
19. Hale, B. G., R. E. Randall, J. Ortin, and D. Jackson. 2008. The multifunctional 34
NS1 protein of influenza A viruses. J Gen Virol 89:2359-76. 35
20. Hale, B. G., J. Steel, B. Manicassamy, R. A. Medina, J. Ye, D. Hickman, A. C. 36
Lowen, D. R. Perez, and A. Garcia-Sastre. 2010. Mutations in the NS1 C-37
terminal tail do not enhance replication or virulence of the 2009 pandemic H1N1 38
influenza A virus. J Gen Virol. 39
21. Hancock, K., V. Veguilla, X. Lu, W. Zhong, E. N. Butler, H. Sun, F. Liu, L. 40
Dong, J. R. DeVos, P. M. Gargiullo, T. L. Brammer, N. J. Cox, T. M. 41 Tumpey, and J. M. Katz. 2009. Cross-reactive antibody responses to the 2009 42
pandemic H1N1 influenza virus. N Engl J Med 361:1945-52. 43
22. Hayman, A., S. Comely, A. Lackenby, S. Murphy, J. McCauley, S. 44
Goodbourn, and W. Barclay. 2006. Variation in the ability of human influenza 45
A viruses to induce and inhibit the IFN-beta pathway. Virology 347:52-64. 46
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
32
23. Hossain, M. J., D. Hickman, and D. R. Perez. 2008. Evidence of expanded host 1
range and mammalian-associated genetic changes in a duck H9N2 influenza virus 2
following adaptation in quail and chickens. PLoS One 3:e3170. 3
24. Itoh, Y., K. Shinya, M. Kiso, T. Watanabe, Y. Sakoda, M. Hatta, Y. 4
Muramoto, D. Tamura, Y. Sakai-Tagawa, T. Noda, S. Sakabe, M. Imai, Y. 5
Hatta, S. Watanabe, C. Li, S. Yamada, K. Fujii, S. Murakami, H. Imai, S. 6
Kakugawa, M. Ito, R. Takano, K. Iwatsuki-Horimoto, M. Shimojima, T. 7
Horimoto, H. Goto, K. Takahashi, A. Makino, H. Ishigaki, M. Nakayama, M. 8
Okamatsu, D. Warshauer, P. A. Shult, R. Saito, H. Suzuki, Y. Furuta, M. 9
Yamashita, K. Mitamura, K. Nakano, M. Nakamura, R. Brockman-10
Schneider, H. Mitamura, M. Yamazaki, N. Sugaya, M. Suresh, M. Ozawa, G. 11 Neumann, J. Gern, H. Kida, K. Ogasawara, and Y. Kawaoka. 2009. In vitro 12
and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature 13
460:1021-5. 14
25. Jackson, D., M. J. Hossain, D. Hickman, D. R. Perez, and R. A. Lamb. 2008. 15
A new influenza virus virulence determinant: the NS1 protein four C-terminal 16
residues modulate pathogenicity. Proc Natl Acad Sci U S A 105:4381-6. 17
26. Jain, S., L. Kamimoto, A. M. Bramley, A. M. Schmitz, S. R. Benoit, J. Louie, 18
D. E. Sugerman, J. K. Druckenmiller, K. A. Ritger, R. Chugh, S. Jasuja, M. 19
Deutscher, S. Chen, J. D. Walker, J. S. Duchin, S. Lett, S. Soliva, E. V. Wells, 20
D. Swerdlow, T. M. Uyeki, A. E. Fiore, S. J. Olsen, A. M. Fry, C. B. Bridges, 21 and L. Finelli. 2009. Hospitalized patients with 2009 H1N1 influenza in the 22
United States, April-June 2009. N Engl J Med 361:1935-44. 23
27. Katze, M. G., and R. M. Krug. 1984. Metabolism and expression of RNA 24
polymerase II transcripts in influenza virus-infected cells. Mol Cell Biol 4:2198-25
206. 26
28. Kochs, G., A. Garcia-Sastre, and L. Martinez-Sobrido. 2007. Multiple anti-27
interferon actions of the influenza A virus NS1 protein. J Virol 81:7011-21. 28
29. Kuo, R. L., and R. M. Krug. 2009. Influenza a virus polymerase is an integral 29
component of the CPSF30-NS1A protein complex in infected cells. J Virol 30
83:1611-6. 31
30. Lange, E., D. Kalthoff, U. Blohm, J. P. Teifke, A. Breithaupt, C. Maresch, E. 32
Starick, S. Fereidouni, B. Hoffmann, T. C. Mettenleiter, M. Beer, and T. W. 33 Vahlenkamp. 2009. Pathogenesis and transmission of the novel swine-origin 34
influenza virus A/H1N1 after experimental infection of pigs. J Gen Virol 35
90:2119-23. 36
31. Li, Y., Z. Y. Chen, W. Wang, C. C. Baker, and R. M. Krug. 2001. The 3'-end-37
processing factor CPSF is required for the splicing of single-intron pre-mRNAs in 38
vivo. RNA 7:920-31. 39
32. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression 40
data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. 41
Methods 25:402-8. 42
33. Long, J. X., D. X. Peng, Y. L. Liu, Y. T. Wu, and X. F. Liu. 2008. Virulence of 43
H5N1 avian influenza virus enhanced by a 15-nucleotide deletion in the viral 44
nonstructural gene. Virus Genes 36:471-8. 45
34. Maines, T. R., A. Jayaraman, J. A. Belser, D. A. Wadford, C. Pappas, H. 46
Zeng, K. M. Gustin, M. B. Pearce, K. Viswanathan, Z. H. Shriver, R. Raman, 47
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
33
N. J. Cox, R. Sasisekharan, J. M. Katz, and T. M. Tumpey. 2009. 1
Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses 2
in ferrets and mice. Science 325:484-7. 3
35. Manicassamy, B., R. A. Medina, R. Hai, T. Tsibane, S. Stertz, E. Nistal-4
Villan, P. Palese, C. F. Basler, and A. Garcia-Sastre. 2010. Protection of mice 5
against lethal challenge with 2009 H1N1 influenza A virus by 1918-like and 6
classical swine H1N1 based vaccines. PLoS Pathog 6:e1000745. 7
36. Mibayashi, M., L. Martinez-Sobrido, Y. M. Loo, W. B. Cardenas, M. Gale, 8
Jr., and A. Garcia-Sastre. 2007. Inhibition of retinoic acid-inducible gene I-9
mediated induction of beta interferon by the NS1 protein of influenza A virus. J 10
Virol 81:514-24. 11
37. Moss, B., O. Elroy-Stein, T. Mizukami, W. A. Alexander, and T. R. Fuerst. 12
1990. Product review. New mammalian expression vectors. Nature 348:91-2. 13
38. Munster, V. J., E. de Wit, J. M. van den Brand, S. Herfst, E. J. Schrauwen, 14
T. M. Bestebroer, D. van de Vijver, C. A. Boucher, M. Koopmans, G. F. 15 Rimmelzwaan, T. Kuiken, A. D. Osterhaus, and R. A. Fouchier. 2009. 16
Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in 17
ferrets. Science 325:481-3. 18
39. Nemeroff, M. E., S. M. Barabino, Y. Li, W. Keller, and R. M. Krug. 1998. 19
Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF 20
and inhibits 3'end formation of cellular pre-mRNAs. Mol Cell 1:991-1000. 21
40. Newby, C. M., L. Sabin, and A. Pekosz. 2007. The RNA binding domain of 22
influenza A virus NS1 protein affects secretion of tumor necrosis factor alpha, 23
interleukin-6, and interferon in primary murine tracheal epithelial cells. J Virol 24
81:9469-80. 25
41. Niwa, H., K. Yamamura, and J. Miyazaki. 1991. Efficient selection for high-26
expression transfectants with a novel eukaryotic vector. Gene 108:193-9. 27
42. Noah, D. L., K. Y. Twu, and R. M. Krug. 2003. Cellular antiviral responses 28
against influenza A virus are countered at the posttranscriptional level by the viral 29
NS1A protein via its binding to a cellular protein required for the 3' end 30
processing of cellular pre-mRNAS. Virology 307:386-95. 31
43. Obenauer, J. C., J. Denson, P. K. Mehta, X. Su, S. Mukatira, D. B. 32
Finkelstein, X. Xu, J. Wang, J. Ma, Y. Fan, K. M. Rakestraw, R. G. Webster, 33 E. Hoffmann, S. Krauss, J. Zheng, Z. Zhang, and C. W. Naeve. 2006. Large-34
scale sequence analysis of avian influenza isolates. Science 311:1576-80. 35
44. Opitz, B., A. Rejaibi, B. Dauber, J. Eckhard, M. Vinzing, B. Schmeck, S. 36
Hippenstiel, N. Suttorp, and T. Wolff. 2007. IFNbeta induction by influenza A 37
virus is mediated by RIG-I which is regulated by the viral NS1 protein. Cell 38
Microbiol 9:930-8. 39
45. Pichlmair, A., O. Schulz, C. P. Tan, T. I. Naslund, P. Liljestrom, F. Weber, 40
and C. Reis e Sousa. 2006. RIG-I-mediated antiviral responses to single-stranded 41
RNA bearing 5'-phosphates. Science 314:997-1001. 42
46. Plotch, S. J., M. Bouloy, I. Ulmanen, and R. M. Krug. 1981. A unique 43
cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs 44
to generate the primers that initiate viral RNA transcription. Cell 23:847-58. 45
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
34
47. Quinlivan, M., D. Zamarin, A. Garcia-Sastre, A. Cullinane, T. Chambers, 1
and P. Palese. 2005. Attenuation of equine influenza viruses through truncations 2
of the NS1 protein. J Virol 79:8431-9. 3
48. Rodriguez, A., A. Perez-Gonzalez, and A. Nieto. 2007. Influenza virus infection 4
causes specific degradation of the largest subunit of cellular RNA polymerase II. J 5
Virol 81:5315-24. 6
49. Seo, S. H., E. Hoffmann, and R. G. Webster. 2002. Lethal H5N1 influenza 7
viruses escape host anti-viral cytokine responses. Nat Med 8:950-4. 8
50. Solorzano, A., R. J. Webby, K. M. Lager, B. H. Janke, A. Garcia-Sastre, and 9
J. A. Richt. 2005. Mutations in the NS1 protein of swine influenza virus impair 10
anti-interferon activity and confer attenuation in pigs. J Virol 79:7535-43. 11
51. Sorrell, E. M., H. Wan, Y. Araya, H. Song, and D. R. Perez. 2009. Minimal 12
molecular constraints for respiratory droplet transmission of an avian-human 13
H9N2 influenza A virus. Proc Natl Acad Sci U S A 106:7565-70. 14
52. Srivastava, B., P. Blazejewska, M. Hessmann, D. Bruder, R. Geffers, S. 15
Mauel, A. D. Gruber, and K. Schughart. 2009. Host genetic background 16
strongly influences the response to influenza a virus infections. PLoS One 17
4:e4857. 18
53. Staeheli, P., P. Dreiding, O. Haller, and J. Lindenmann. 1985. Polyclonal and 19
monoclonal antibodies to the interferon-inducible protein Mx of influenza virus-20
resistant mice. J Biol Chem 260:1821-5. 21
54. Swayne, D. E., M. Pantin-Jackwood, D. Kapczynski, E. Spackman, and D. L. 22
Suarez. 2009. Susceptibility of poultry to pandemic (H1N1) 2009 Virus. Emerg 23
Infect Dis 15:2061-3. 24
55. Talon, J., C. M. Horvath, R. Polley, C. F. Basler, T. Muster, P. Palese, and A. 25
Garcia-Sastre. 2000. Activation of interferon regulatory factor 3 is inhibited by 26
the influenza A virus NS1 protein. J Virol 74:7989-96. 27
56. Twu, K. Y., R. L. Kuo, J. Marklund, and R. M. Krug. 2007. The H5N1 28
influenza virus NS genes selected after 1998 enhance virus replication in 29
mammalian cells. J Virol 81:8112-21. 30
57. Twu, K. Y., D. L. Noah, P. Rao, R. L. Kuo, and R. M. Krug. 2006. The 31
CPSF30 binding site on the NS1A protein of influenza A virus is a potential 32
antiviral target. J Virol 80:3957-65. 33
58. Vreede, F. T., A. Y. Chan, J. Sharps, and E. Fodor. 2009. Mechanisms and 34
functional implications of the degradation of host RNA polymerase II in influenza 35
virus infected cells. Virology. 36
59. Wang, X., M. Li, H. Zheng, T. Muster, P. Palese, A. A. Beg, and A. Garcia-37
Sastre. 2000. Influenza A virus NS1 protein prevents activation of NF-kappaB 38
and induction of alpha/beta interferon. J Virol 74:11566-73. 39
60. Yoneyama, M., W. Suhara, Y. Fukuhara, M. Fukuda, E. Nishida, and T. 40
Fujita. 1998. Direct triggering of the type I interferon system by virus infection: 41
activation of a transcription factor complex containing IRF-3 and CBP/p300. 42
EMBO J 17:1087-95. 43
44
45
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
35
FIGURE LEGENDS 1
2
FIGURE 1. Inhibition of IFNββββ induction by different influenza A virus NS1 3
proteins. 293T cells were co-transfected for 16 h with a pCAGGS expression plasmid 4
encoding the indicated NS1 protein (or GST) together with a FF-luciferase IFNβ-5
promoter reporter plasmid (p125Luc). After infection with SeV for a further 12 h 6
luciferase (FF-Luc) activity was determined. Zoomed inset highlights differences 7
between NS1 proteins. Results represent the means and standard deviations of triplicate 8
values (normalized to GST + SeV) obtained in a single experiment, and are representative 9
of two independent experiments. 10
11
FIGURE 2. The Cal/09 NS1 protein is unable to block general gene expression in 12
human or swine cells. Human 293T (A), or swine PK-15 (C) cells were co-transfected 13
with a pCAGGS expression plasmid encoding the indicated NS1 protein (or GST) 14
together with a constitutively active Renilla-luciferase plasmid. Luciferase activity was 15
determined 24 h post-transfection. Results show the means and standard deviations of 16
triplicate values [normalized to GST (A), or PR/34 NS1 (C)] obtained in a single 17
experiment, and are representative of two independent experiments. (B) Western blot 18
analysis of lysates from (A). (D) Western blot analysis of lysates from (C). NS1 and GST 19
were detected using a polyclonal rabbit anti-serum raised against a fusion protein of GST 20
and the N-terminal RNA binding domain of NS1. Tubulin acted as a loading control. 21
Molecular weight markers (kDa) are indicated to the right. 22
23
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
36
FIGURE 3. Residues at the interface of the NS1:CPSF30 complex. (A) Amino-acid 1
sequence alignment of the NS1 effector domains (ED, residues 85-203) from PR/34, 2
BM/18, Tx/91, Sw/Tx/98, and Cal/09. Identity (*) and level of similarity (: or .) are 3
indicated. Gray shading highlights residues of NS1 previously shown experimentally to 4
be important for CPSF30 binding. Black boxes highlight residues common to PR/34, 5
BM/18, and Tx/91 NS1-EDs (‘human-like’), but different in Sw/Tx/98 and Cal/09 NS1-6
EDs (‘swine-like’). Residues 108, 125, and 189 (which are the only residues unique to 7
Sw/Tx/98 and Cal/09 NS1 proteins at the NS1:CPSF30 interface, see below) are labeled. 8
(B) Structural representation of NS1 in complex with the F2F3 fragment of CPSF30. 9
Monomeric NS1-ED is shown in cartoon format (green), whilst the CPSF30-F2F3 10
fragment is shown in surface representation (blue). Unique residues of Sw/Tx/98 and 11
Cal/09 NS1-EDs compared to PR/34, BM/18, and Tx/91 are highlighted with black 12
spheres. (C) Close-up view of the NS1:CPSF30 interface with residues different in 13
Sw/Tx/98 and Cal/09 NS1-EDs highlighted in black (sticks). Panels B and C were 14
generated using MacPyMol [PDB ID: 2RHK (8)]. 15
16
FIGURE 4. Three amino-acid substitutions restore ability of the Cal/09 NS1 protein 17
to block general gene expression in human and swine cells. Human 293T (A), or 18
swine PK-15 (C) cells were co-transfected with a pCAGGS expression plasmid encoding 19
the indicated WT NS1 protein or the Cal/09 mutants: R108K, E125D, G189D, 20
R108K/G189D (108/189), or R108K/E125D/G189D (TripleMut); together with a 21
constitutively active Renilla-luciferase plasmid. 24 h post-transfection luciferase activity 22
was determined. Results show the means and standard deviations of triplicate values 23
(normalized to WT Cal/09 NS1) obtained in a single experiment, and are representative 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
37
of two independent experiments. (B and D) Western blot analysis of lysates from (A) and 1
(C), respectively. NS1 and tubulin were detected as for FIG. 2. 2
3
FIGURE 5. Interaction of CPSF30 with WT Cal/09 NS1 or TripleMut Cal/09 NS1. 4
FLAG-tagged CPSF30 was expressed in 293T cells (CPSF30), mixed with in vitro 5
synthesized 35
S-methionine labeled NS1 (WT Cal/09, TripleMut Cal/09, or HA-tagged 6
Tx/91), and immunoprecipitated using anti-FLAG resin. 293T lysates without FLAG-7
tagged CPSF30 (Mock) acted as a negative control. Following SDS-PAGE, precipitated 8
proteins were detected by Coomassie blue staining (IgG), Western blot (FLAG-tagged 9
CPSF30), or phosphorimaging (35
S-methionine labeled NS1). Immunoprecipitations were 10
performed using 500 mM NaCl (A, ‘In’ denotes 10% input) or 200 mM NaCl (B, ‘In’ 11
denotes 1% input). (C) Ability of WT Cal/09 NS1 or TripleMut Cal/09 NS1 to bind 12
synthetic dsRNA. Lysates from A549 cells infected with rCal/09 WT or rCal/09 13
TripleMut (MOI of 5 PFU/cell) were precipitated with pI:C-Sepharose (pI:C) or 14
Sepharose only (-). Following SDS-PAGE, NS1 proteins were detected by Western blot. 15
Molecular weight markers (kDa) are indicated to the right. 16
17
FIGURE 6. In vitro characterization of the rCal/09 TripleMut virus. (A) Multicycle 18
growth analysis of rCal/09 WT and non-recombinant Cal/09 WT viruses in primary 19
differentiated human airway epithelial cells. (B) Plaque phenotype of rCal/09 WT and 20
rCal/09 TripleMut viruses in MDCK cells. (C) Western blot determination of viral NP 21
and NS1 protein expression in MDCK cells infected for 12 h with the rCal/09 WT or 22
rCal/09 TripleMut (TM) viruses (MOI of 5 PFU/cell). Molecular weight markers (kDa) 23
are indicated to the right. Multicycle growth analysis of rCal/09 WT and rCal/09 24
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
38
TripleMut viruses in primary differentiated human airway epithelial cells (D), and the 1
swine PK-15 cell-line (E). Data points for growth curves show mean values (n=3) and 2
error bars represent standard deviation. 3
4
FIGURE 7. Ability of rCal/09 TripleMut virus to limit expression of IFNββββ and IFN-5
stimulated genes during infection. Primary undifferentiated human airway epithelial 6
cells were infected at an MOI of 5 PFU/cell with rCal/09 WT or rCal/09 TripleMut 7
viruses. (A) Infectious titers determined by plaque assay in supernatants after 24 h. (B-F) 8
Cells were lysed at the times indicated and total RNA extracted. Following reverse 9
transcription using oligo(dT) the levels of viral NA mRNA (B), IFNβ mRNA (C), OAS 10
mRNA (D), MxA mRNA (E), and IP10 mRNA (F) were quantified in triplicate by 11
qPCR. Values were averaged and normalized to 18S RNA. Mean fold-induction relative 12
to mRNA levels in mock-infected cells is shown. Error bars represent standard deviation. 13
Means and standard deviations were calculated from biological triplicates. 14
15
FIGURE 8. Replication and pathogenicity of the rCal/09 TripleMut virus in mice. 7-16
week old DBA/2J mice were infected intranasally with 105 PFU of rCal/09 WT or 17
rCal/09 TripleMut viruses. PBS-treated mice acted as a negative control. (A) Body 18
weights were determined daily. Data show mean body weight (n=5). Error bars represent 19
standard deviations. Significance was determined using a two-tailed Student’s t-test (**, 20
p < 0.01, *, p < 0.05). (B) Lung titers were determined on days 2, 4, and 7 post-infection 21
from three DBA/2J mice infected in parallel with 104 PFU of rCal/09 WT or rCal/09 22
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
39
TripleMut viruses. Bars represent mean values. (C) Murine IFNβ mRNA was quantified 1
from the lung homogenates used in (B) by qPCR as for FIG. 7. 2
3
FIGURE 9. Replication and transmission dynamics of the rCal/09 TripleMut virus 4
in ferrets. 6-8 month old female Fitch ferrets were infected intranasally with 106 TCID50 5
of rCal/09 WT or rCal/09 TripleMut viruses. (A) Viral titers in nasal turbinates, trachea 6
and lungs were determined on day 5 post-infection from two infected ferrets. Bars 7
represent mean values. (B-C) Nasal wash titers for rCal/09 WT (B) and rCal/09 8
TripleMut (C) viruses were determined from infected ferrets (n=2), direct contact ferrets 9
(n=2), and aerosol (large and small respiratory droplet) contact ferrets (n=2) on the days 10
indicated. Bars (dark and light gray) represent the raw results of two independent 11
experiments. To limit the number of animals used, the rCal/09 WT-infected ferret data 12
shown are the same as previously described (20). All experiments were performed in 13
parallel to allow fair comparison. 14
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
40
TABLE 1. Species- and subtype- specific identity of NS1 residues involved in 1
CPSF30 binding. 2
% of NS1 sequences with specific residues at stated positionsa,b
103 106 108 125 189
Fc
Y X
d M
c X
d K
c R
X
d
D
c E
X
d D
c G
X
d
Aviane
H1 71 29 0 100 0 63 37 0 90 8 2 91 9 0
H3 57 43 0 100 0 52 48 0 95 5 0 95 5 0
H6 45 23 33 68 33 77 24 0 95 0 5 99 1 0
H9 26 3 71 24 76 97 3 0 98 0 2 99 0 1
Human
e
H1 98 0 2 98 2 99 1 0 95 5 0 97 2 1
H3 100 0 0 100 0 100 0 0 6 94 0 100 0 0
2009
H1N1 98 0 2 100 0 0 100 0 0 100 0 0 100 0
Swinee
H1 97 0 3 98 2 34 63 3 23 71 6 22 74 4
H3 97 0 3 95 5 62 36 2 19 65 16 62 36 2
3
aPercentage of NS1 sequences with certain amino-acid residues located at positions 103, 4
106, 108, 125, and 189. bAll sequences are derived from publicly-available databases (as 5
of November 2009, or April 2010 for the 2009 H1N1 virus) and were aligned prior to 6
analysis. cBold, underlined residues contribute to stable CPSF30 binding.
dX represents 7
‘other’ amino-acids. eThe number of sequences for each group was as follows: Avian 8
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
41
(H1=99, H3=293, H6=400, H9=632), Human (H1=898, H3=2167, 2009 H1N1=1568), 1
and Swine (H1=302, H3=119). Values are rounded to the nearest whole percentage point. 2
on February 2, 2019 by guest
http://jvi.asm.org/
Dow
nloaded from
Recommended
