Inefficient control of host gene expression by the 2009 pandemic. H1N1 influenza A virus NS1 protein

Transcription

1 JVI Accepts, published online ahead of print on May 0 J. Virol. doi:./jvi Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Inefficient control of host gene expression by the 00 pandemic H1N1 influenza A virus NS1 protein Benjamin G. Hale 1, John Steel 1, Rafael A. Medina 1,, Balaji Manicassamy 1,, Jianqiang Ye, Danielle Hickman, Rong Hai 1, Mirco Schmolke 1, Anice C. Lowen 1, Daniel R. Perez & Adolfo García-Sastre 1,,,* 1 Department of Microbiology, Department of Medicine, Division of Infectious Diseases, Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 0, USA. Department of Veterinary Medicine, University of Maryland, College Park, MD 0, USA. * Corresponding author. Mailing address: Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 0. Phone: (1) 1-. Fax: (1) adolfo.garcia-sastre@mssm.edu. Running Title: 00 Pandemic H1N1 NS1 Protein and CPSF0. Words in abstract:. Words in main text:,. Number of figures and tables: figures + 1 table. Number of references: 0. Downloaded from on November, 01 by guest 1

2 ABSTRACT In 00, a novel swine-origin H1N1 influenza A virus emerged. Here, we characterize the multifunctional NS1 protein of this human pandemic virus in order to understand factors that may contribute towards replication efficiency or pathogenicity. Although the 00 H1N1 virus NS1 protein (00/NS1) is an effective interferon-antagonist, we found that this NS1 (unlike those of previous human-adapted influenza A viruses) is unable to block general host gene expression in human or swine cells. This property could be restored in 00/NS1 by substituting R, E1 and G1 for residues corresponding to human virus consensus. Mechanistically, these previously undescribed mutations acted by increasing binding of 00/NS1 to the cellular pre-mrna processing protein CPSF0. A recombinant 00 H1N1 influenza A virus (A/California/0/0) expressing NS1 with these gain-of-function substitutions was more efficient than wild-type at antagonizing host innate immune responses in primary human epithelial cells. However, such mutations had no significant effect on virus replication in either human or swine tissueculture substrates. Surprisingly, in a mouse model of pathogenicity, the mutant virus appeared to cause less morbidity, and was cleared faster, than wild-type. The mutant virus also demonstrated reduced titers in the upper respiratory tracts of ferrets, however contact and aerosol transmissibility of the virus were unaffected. Our data highlight a potential human adaptation of NS1 that seems absent in classically-derived swine-origin influenza A viruses, including the 00 H1N1 virus. We discuss the impact that natural future gain of this NS1 function may have on the new pandemic virus in humans. Downloaded from on November, 01 by guest

3 INTRODUCTION In April 00, an antigenically distinct swine-origin H1N1 influenza A virus was detected in humans (1). This virus has since spread efficiently around the world, leading to the declaration of a global pandemic by the World Health Organization on th June 00. Although infection with the virus is generally associated with a mild self-limiting influenza-like disease in the majority of people, the young and those with certain underlying conditions (including asthma, diabetes, heart/lung problems, morbid obesity, and pregnancy) seem at greater risk of severe disease progression (). Lack of significant pre-existing immunity to the novel virus in those <0 years old (1, ) may account for the observation that younger people are more susceptible to this virus (). Sequence analysis of the 00 pandemic H1N1 influenza A virus genome has failed to identify any previously recognized virulence markers (1, 1, ). Nevertheless, animal studies have indicated that the 00 H1N1 virus is slightly more pathogenic than contemporary human seasonal H1N1 viruses (,, ). In ferrets, the pandemic H1N1 virus replicated to higher titers than seasonal H1N1 viruses in the upper respiratory tract (,, ) and, unusually, could be detected deeper in the lungs (, ) and intestinal tracts () of infected animals. In a mouse model, the 00 H1N1 virus also replicated more efficiently, and caused greater morbidity and mortality, than seasonal influenza viruses (, ). Furthermore, mice infected with the 00 H1N1 virus produced higher levels of several pro-inflammatory cytokines and chemokines, including IFNγ, IL, IL, IL1, MIP1α, MIP1β, and RANTES (). Similar data for the 00 H1N1 virus with Downloaded from on November, 01 by guest

4 respect to its efficient replication and significant induction of cytokines (together with severe lung pathology) have also been obtained from experimentally infected cynomolgus macaques (). As might be expected given its swine origin, the 00 H1N1 virus replicates well in pigs, although whether the virus causes significant disease in this host is unclear (, 0). In experimental infections of avian species the 00 H1N1 virus is unable to replicate efficiently or cause disease (, ). Influenza A virus virulence is a polygenic trait. Multiple unidentified molecular determinants are likely responsible for the ability of the 00 H1N1 influenza A virus to cause increased disease severity in certain host species. Here, we have focused on the non-structural (NS1) protein of the 00 H1N1 virus. NS1 performs a number of diverse roles in infected cells [reviewed in (1)]. The best-described function of NS1 is its ability to limit host-cell cytokine production [e.g. interferon (IFN)] during infection (1, 0,, ). This is due to at least two independent NS1 activities (): (i) the pre-transcriptional inhibition of RIG-I [through sequestration of this RNA helicase and its activating ligand (1,,, ), or inhibition of TRIM-mediated RIG-I ubiquitination ()], and (ii) the global post-transcriptional inhibition of CPSF0-mediated cellular pre-mrna processing (, ). Both of these activities appear to contribute to the replication efficiency of human influenza A viruses, and both likely play roles in determining virulence (,, 1, ). There are recently documented examples of non-human adapted virus strains (both laboratory and naturally-occurring) that encode NS1 proteins lacking the ability to inhibit CPSF0 (,, ). The commonly used egg- and mouse- adapted human-derived Downloaded from on November, 01 by guest

5 influenza virus A/Puerto Rico// (PR/) NS1 protein has amino-acid substitutions at residues and, and is consequently unable to bind CPSF0 or block general gene expression (, ). The NS1 proteins of two avian-derived influenza A viruses (the highly-pathogenic A/Hong Kong// [HN1] strain that was transmitted to humans in 1, and its presumed precursor A/Teal/Hong Kong/W1/ [HN1]) are additionally unable to inhibit CPSF0 in either human or avian cells, an intrinsic defect in NS1 function that also maps to substitutions at positions and (). Sequence analysis indicates that such substitutions are common amongst avian H and H HA subtypes, and it has been suggested that these viruses may tolerate (or even be selected for) inefficient CPSF0 binding (). Furthermore, NS1:CPSF0-destabilizing substitutions at positions or have been reported to arise when viruses are adapted to replicate well in certain new host species [e.g. duck to quail (), and human to mouse ()]. The biological reasons and apparent selection pressures required for some viral strains to modulate their NS1:CPSF0 binding affinities in particular hosts are unclear. The ability of swine-derived influenza virus NS1 proteins to function efficiently in human cells has not yet been fully tested. Galvanized by the worldwide spread of swine-origin H1N1 influenza A virus in humans, we assessed the effectiveness of this pandemic virus gene product to antagonize the human innate immune response. Surprisingly, we found that the 00/NS1 protein is unable to block general host gene expression in both human and swine cells. This is a consequence of previously undescribed amino-acid changes in NS1 that reduce its binding to CPSF0. Such changes appear to have occurred as soon as the parental virus from which this NS1 is derived (likely the 11 pandemic H1N1 influenza A virus) was introduced into pigs and established the current classical swine Downloaded from on November, 01 by guest

6 H1N1 lineage. Here, we characterize the functional consequences of restoring this lost function in the 00 pandemic H1N1 virus with specific regard to host innate immune suppression, virus pathogenicity and virus transmissibility in mouse and ferret animal models. Downloaded from on November, 01 by guest

7 MATERIALS AND METHODS Cells. T (human), A (human), MDCK (canine), and PK-1 (swine) cells were purchased from the American Type Culture Collection (ATCC, VA, USA) and maintained in DMEM supplemented with % FBS, 0 units/ml of penicillin, and 0 µg/ml of streptomycin (GIBCO Life Technologies, CA, USA). Cryopreserved primary human tracheobronchial epithelial cells (HTBEs; Clonetics, Lonza, Walkersville, MD, USA) were cultivated as previously described (1). Briefly, after thawing, cells were passaged twice in bronchial epithelial growth medium (BEGM; Clonetics, Lonza, Walkersville, MD, USA) supplemented with retinoic acid. For full differentiation and generation of an air-liquid interface, cells were seeded onto collagen-coated 1 mm Transwell-Clear Permeable filters (0. µm pores, Corning Inc., MA, USA; collagen I from human placenta, Sigma-Aldrich, MO, USA) at a density of x cells/filter. Cells were submerged for 1 week in a 1:1 mixture of DMEM and BEGM (Gray s medium) containing necessary supplements and growth factors. At confluence, medium was removed from the apical surface, and cells were maintained at the air-liquid interface for at least weeks. Medium was replaced every second day when cells were submerged, and daily when cells were incubated at the air-liquid interface. Plasmids. Mammalian expression constructs for untagged NS1 under control of the chicken β-actin promoter [pcaggs vector (1)] have been described previously for A/Puerto Rico// [PR/ ()], A/Texas//1 [Tx/1 ()], A/Brevig Mission/1/1 [BM/1 ()], and A/Swine/Texas/1-/ [Sw/Tx/ ()]. To generate an expression Downloaded from on November, 01 by guest

8 construct for A/California/0/0 NS1 (Cal/0), cdna encoding this NS1 protein was ligated into pcaggs using the EcoRI and NheI restriction sites. Four-primer overlap PCR was used to introduce site-directed point mutations into the NS1-encoding cdnas. All NS1-encoding cdnas also contained silent mutations in the splice acceptor site in order to prevent expression of NS/NEP (). A pcaggs vector expressing GST was kindly provided by Luis Martínez-Sobrido (University of Rochester Medical Center, NY, USA). A pcaggs vector expressing C-terminal FLAG-tagged human CPSF0 has been described previously (). T-driven expression constructs for wild-type or mutant Cal/0 NS1 proteins were generated by ligating the appropriate PCR-amplified cdnas between the NcoI and BamHI restriction sites of ptm1 (). The pcdna plasmid encoding HA-tagged Tx/1 NS1 has been described previously (). The reporter plasmid carrying the firefly luciferase (FF-Luc) gene under control of the IFNβ promoter (p1luc) was kindly provided by Takashi Fujita (Kyoto University, Japan) (0). The reporter plasmid carrying the Renilla luciferase gene (REN-Luc) under control of the constitutively active HSV-TK promoter (prl-tk) was purchased from Promega, WI, USA. The identity of each construct generated or used was confirmed by sequencing. Viruses. Stocks of Sendai virus (SeV; Cantell strain) were propagated in -day old embryonated chicken eggs. The reference 00 pandemic H1N1 influenza A virus isolate, A/California/0/0 (Cal/0), was obtained from the Centers for Disease Control and Prevention (CDC Atlanta, GA, USA), and propagated in MDCK cells. Recombinant rcal/0 (wild-type, WT) was rescued according to previously reported protocols (, 1, ), with minor modifications. Briefly, bi-directional pdz-based Cal/0 RNA Downloaded from on November, 01 by guest

9 expression plasmids (HA, NA, M, NP, PA, PB1, and PB) were co-transfected with a ppoli-based Cal/0 NS vrna expression plasmid into T cells. At h posttransfection, growth medium was replaced with serum-free medium containing 1 µg/ml TPCK-treated trypsin (Sigma-Aldrich, MO, USA) and MDCK cells were added to the culture. At h post-transfection, rescue supernatant was subjected to plaque assay on MDCK cells in order to obtain clonal isolates. For rescue of the rcal/0 TripleMut virus (NS1 amino-acid substitutions RK, E1D, and G1D), site-directed mutagenesis was used to introduce the required mutations into ppoli-cal/0-ns using the QuikChange II Site Directed Mutagenesis Kit (Stratagene, TX, USA). The identity of the construct was confirmed by DNA sequencing. Rescue of the mutant virus was performed as described for wild-type. Rescued viruses were propagated in MDCK cells, and the genotypes of the rcal/0 WT and TripleMut viruses were confirmed by RT-PCR and sequencing of the entire NS segments. As required by the Mount Sinai School of Medicine biosafety committee, work involving Cal/0 viruses was carried out in either a USDA and CDC-approved BSL+ containment laboratory, or a dedicated BSL laboratory with personnel adhering to BSL working practices. Reporter assays. For analysis of IFNβ promoter activation, T cells in 1-well plates were transfected with ng of p1luc and µg of the indicated NS1 (or GST) expression plasmid using FuGENE (Roche, WI, USA). After 1 h, the cells were infected with approximately 1 PFU/cell of SeV for 1 h. Cells were harvested and lysed in 00 µl of passive lysis buffer (Promega, WI, USA), and FF-Luc activity was determined using a luminometer. To measure gene expression under control of the Downloaded from on November, 01 by guest

10 constitutively active HSV-TK promoter, T or PK-1 cells in 1-well plates were cotransfected with 0 ng of prl-tk and µg of the indicated NS1 (or GST) constructs using FuGENE (Roche, WI, USA). Expression of REN-Luc activity was measured h post-transfection as directed by the manufacturer (Promega, WI, USA). All transfections were carried out in triplicate, and experiments were independently repeated at least twice. SDS-PAGE, Western blot and immunoprecipitations. Cells were lysed in disruption buffer ( M urea, M β-mercaptoethanol, % SDS), sonicated to shear nucleic acids, and boiled for min prior to polypeptide separation by SDS-PAGE on -1% Tris-HCl gradient gels (Bio-Rad Laboratories, CA, USA). Proteins were detected either by Coomassie blue staining or by Western blot analysis following transfer to polyvinylidene difluoride (PVDF) membranes. The rabbit anti-tubulin and mouse anti-flag antibodies were from Sigma-Aldrich, MO, USA. Rabbit polyclonal anti-serum to detect NP, and rabbit polyclonal anti-serum raised against a GST-NS1(RBD) fusion protein (that detects both GST and NS1) have been described previously (0). To assess the interaction of different Cal/0 NS1 constructs with FLAG-tagged CPSF0, S-methionine labeled NS1 proteins were synthesized in vitro from ptm1-cal/0-ns1 expression plasmids using a TNT transcription/translation kit (Promega, WI, USA). T cells transiently-expressing FLAG-CPSF0 were lysed in 0 mm Tris-HCl (ph.), 00 mm NaCl, mm EDTA, and 0.% NP-0, supplemented with a complete Mini protease inhibitor cocktail (Roche, IN, USA). Following sonication, cleared cell lysates expressing FLAG-CPSF0 (or not) were incubated for h at C with the radiolabeled NS1 proteins and µl of anti-flag affinity resin (Sigma-Aldrich, MO, USA). After extensive washing, precipitated proteins Downloaded from on November, 01 by guest

11 were dissociated from the resin using disruption buffer and analyzed by SDS-PAGE followed by Coomassie blue staining or Western blot. The S-methionine labeled NS1 proteins were detected by phosphorimager analysis Poly(rI):poly(rC)-Sepharose co-precipitations. A cells were infected with the appropriate virus at an MOI of PFU/cell, and lysates were prepared after 1 h in 0 mm Tris-HCl (ph.), 00 mm NaCl, mm EDTA, and 0.% NP-0, supplemented with a complete Mini protease inhibitor cocktail (Roche, IN, USA). Following sonication, clarified lysates were incubated for h at C with poly(ri):poly(rc) (pi:c) Sepharose [generated as described previously ()] or Sepharose only. After extensive washing, precipitated proteins were dissociated from the resin using disruption buffer and analyzed by SDS-PAGE followed by Western blot. Virus growth analysis. Cultures of primary differentiated HTBEs were washed extensively with PBS (to remove accumulated mucus), and infected in triplicate with the appropriate virus at an MOI of PFU/cell (in the absence of exogenous TPCKtreated trypsin). Following infection, cells were maintained at the air-liquid interface. Culture supernatants were harvested at various times post-infection by addition of PBS to the apical surface of the cells for 0 min. Growth analysis in PK-1 cells was essentially the same, albeit no air-liquid interface was established and cells were maintained throughout in Opti-MEM (GIBCO Life Technologies, CA, USA) supplemented with % bovine serum albumin and 1 µg/ml TPCK-treated trypsin. Virus titers were determined by plaque assay on MDCK cells. Plaques were visualized either by crystal violet staining, Downloaded from on November, 01 by guest

12 or by immunostaining with a rabbit polyclonal anti-serum raised against whole- inactivated virus (A/Puerto Rico//) (0) Quantitative real-time PCR (qpcr). Undifferentiated HTBEs in 1-well plates were infected in triplicate (or mock) with virus at an MOI of PFU/cell. At various times postinfection, cells were lysed and total RNA extracted using the RNeasy Mini Kit (Qiagen, CA, USA) according to the manufacturer s instructions. All samples were subjected to DNAse treatment (Qiagen, CA, USA). For reverse transcription, µg of total RNA and 0. µg oligo(dt) primer (in a total volume of 1. µl) was heated for min at C. After addition of enzyme reaction buffer, 0 units RiboLock RNase inhibitor, 0 nmol dntps, and 00 units RevertAid M-MuLV reverse transcriptase (Fermentas Inc., MD, USA), samples were heated at C for 1 h. Reverse transcriptase activity was subsequently inactivated at 0 C for min. The resulting cdna product acted as the template for qpcr using the Brilliant QPCR SYBR Green Mastermix system (Stratagene, TX. USA) and a Roche Light Cycler 0. The assay was performed according to the manufacturers instructions, and the fragment of interest was amplified in 0 cycles. Primer sequences used are available upon request. Samples were tested in triplicate, and cdna levels normalized to 1S RNA levels. Fold-induction over the mock-infected samples was calculated according to the - CT -method (). Mouse experiments. All mouse procedures were performed in accordance with the Institutional Animal Care and Use Committee guidelines of Mount Sinai School of Medicine, and were carried out in a BSL containment laboratory. -week old female Downloaded from on November, 01 by guest 1

13 congenic B.AG-Mx1 mice () carrying intact Mx1 alleles on the CBL/ background (kindly provided by Peter Staeheli, University of Freiburg, Germany and bred locally) or DBA/J mice (The Jackson Laboratory, ME USA) were anaesthetized using ketaminexylazine and intranasally infected with the indicated dose of virus diluted in 0 µl of PBS. Body weights were subsequently measured daily for weeks post-infection. Mice showing more than % weight loss were considered to have reached the experimental endpoint and were humanely euthanized. Lungs were excised from euthanized mice and homogenized in 1 ml PBS supplemented with 0.% bovine serum albumin and penicillin/streptomycin using a mechanical homogenizer (MP Biochemicals, OH, USA). After centrifugation (,000 x g, min, C), resulting supernatants were used to determine viral titer, and pelleted debris was used for RNA extraction using TRIzol (Invitrogen, CA, USA). Ferret experiments. All ferret procedures were performed in accordance with the Institutional Animal Care and Use Committee guidelines of the University of Maryland, and were carried out in a USDA-approved BSL+ containment laboratory. The basic transmission study scheme consisted of duplicate groups of three - month old female Fitch ferrets (Triple F Farms, PA USA): one infected (intranasal with TCID 0 of virus in PBS, 00 µl per nostril), one direct contact and one respiratory droplet contact. Nasal washes were collected daily. For tissue collection, infected ferrets were humanely euthanized on day post-infection, and nasal turbinate, trachea and lung samples were collected. All experimental details were essentially as described previously (1), and virus samples from nasal washes and tissues were titrated on MDCK cells. Downloaded from on November, 01 by guest 1

14 RESULTS IFN-antagonistic properties of the 00 H1N1 virus NS1 protein. We tested the ability of the swine-origin 00 pandemic H1N1 virus NS1 protein to limit production of IFNβ in human cells, and compared it with a panel of other NS1 proteins known to inhibit (or not (, )) general gene expression by binding CPSF0. T cells were transfected with a firefly-luciferase IFNβ promoter reporter construct together with expression plasmids for the NS1 proteins of A/Puerto Rico// [PR/], A/Texas//1 [Tx/1], A/Brevig Mission/1/11 [BM/1], A/Swine/Texas/1-/ [Sw/Tx/], or a representative isolate of the 00 pandemic H1N1 influenza A virus, A/California/0/0 [Cal/0]. A GST expression plasmid served as a negative control. Sixteen hours posttransfection, cells were infected with SeV for a further 1 h prior to analysis of FFluciferase activity. As shown in FIG. 1, SeV infection induced robust amounts of IFNβ promoter-driven FF-luciferase activity in GST-expressing cells (set to 0%), but not in cells expressing any of the NS1 proteins (< %), including the NS1 protein of the 00 pandemic H1N1 influenza A virus (Cal/0). Despite this clear inhibition by all NS1 proteins tested, there was a minor (yet statistically significant) difference: whilst both the NS1 proteins of Tx/1 and BM/1 completely blocked induction of FF-luciferase activity, the NS1 proteins of PR/, Sw/Tx/, and Cal/0 were unable to inhibit a small fraction of activity (i.e. -%; FIG. 1 inset). Given that a major phenotypic difference between the NS1 proteins of PR/ and Tx/1 is their ability to bind CPSF0 and thus to limit gene expression (), we postulated that the incomplete block demonstrated by both Sw/Tx/ and Cal/0 NS1 proteins was, like PR/, due to an inability to inhibit Downloaded from on November, 01 by guest 1

15 CPSF0. We suspect that pre-transcriptional block of TRIM/RIG-I by NS1 (, 1,,,,, ) accounts for the majority of IFNβ-promoter inhibition in our assay The 00 H1N1 virus NS1 protein is unable to block general host gene expression in either human or swine cells. To formally test whether the Cal/0 NS1 protein is unable to block general host gene expression, we co-transfected a constitutively-active Renillaluciferase reporter construct (prl-tk) with each of the indicated NS1 constructs and measured total Renilla-luciferase activity h later. Previous data from our laboratory have demonstrated a clear link between the ability of NS1 proteins to bind CPSF0 and their ability to block Renilla-luciferase reporter activity in this assay (). As shown in FIG. A, only the NS1 proteins of Tx/1 and BM/1 efficiently inhibited Renillaluciferase activity in human T cells, whilst the NS1 proteins of PR/ and the swinederived Sw/Tx/ and Cal/0 (like GST) were largely deficient in this function. We also analyzed the total amounts of GST or NS1 protein in these transfected cell lysates. As expected, the NS1 proteins that efficiently blocked Renilla-luciferase activity (Tx/1 and BM/1) were barely detectable (FIG. B), presumably because the small amount of NS1 protein made is sufficient to limit maturation of further RNA polymerase II transcripts from its own transfected plasmid, as well as from the Renilla-luciferase plasmid. In contrast, the NS1 proteins of PR/, Sw/Tx/, and Cal/0 expressed well (FIG. B), probably as they are unable to block their own expression. These observations are entirely consistent with previous results obtained comparing NS1 proteins able (or not) to bind and inhibit CPSF0 (). Downloaded from on November, 01 by guest 1

16 Given that both Sw/Tx/ and Cal/0 NS1 proteins are derived from swine-origin viruses, and therefore may specifically not function in human cells, we tested whether these NS1 proteins were better adapted to block general gene expression in swine cells. Thus, we also performed the Renilla-luciferase reporter activity assays in transfected PK- 1 cells, a porcine kidney epithelial cell-line. However, as shown in FIGs. C and D, the results obtained were essentially identical to those obtained using a human cell-line: Tx/1 and BM/1 NS1 proteins inhibited gene expression (both of the Renilla-luciferase reporter and themselves), whilst the NS1 proteins of PR/, Sw/Tx/, and Cal/0 did not. This suggests that the inability of swine-origin influenza virus NS1 proteins to block gene expression is not restricted to non-swine cells. Furthermore, in agreement with the hypothesis that failure of these NS1 proteins to block gene expression is related to their inability to bind CPSF0, the CPSF0 amino-acid sequences between human and swine are.% identical (/ residues, G. Pisanelli and A. G-S, unpublished). Substitution of residues, 1, and 1 in the 00 H1N1 virus NS1 protein restore its ability to inhibit general gene expression and to bind CPSF0. The interaction between NS1 and CPSF0 is primarily mediated by the NS1 effector domain (ED, residues -0) and two zinc-fingers of CPSF0 (the FF region) (, ). Based on the assumption that the NS1 phenotypes were due to their differential abilities to bind CPSF0, we aligned the amino-acid sequences of the NS1-EDs used in this study (FIG. A). Overall, the EDs are highly conserved, and the few amino-acid residues of NS1 that have been experimentally implicated in CPSF0 binding to date (e.g.,, and 1-1 (,, 1,, ), FIG. A, gray shading) are no different between the NS1 proteins of BM/1 or Tx/1 (which block gene expression) and Sw/Tx/ or Cal/0 Downloaded from on November, 01 by guest 1

17 (which lack this function). Furthermore, residues and (which determine lack of CPSF0 binding for PR/ NS1) are also human binding consensus for the Cal/0 NS1 protein. In fact, there are only nine amino-acid positions in the ED that consistently differ between the swine-origin viruses and all the human-like viruses (FIG. A, black boxes). Notably, three of these residues are located at the direct interface between NS1 and CPSF0 ():, 1, and 1 (FIGs. B and C), although none of these NS1 residues have previously been experimentally shown to contribute towards CPSF0 binding. We constructed Cal/0 NS1 expression plasmids with each of the following single aminoacid substitutions: RK, E1D, and G1D (Cal/0 residues were changed to human influenza A virus consensus), and tested their ability to block general gene expression in our Renilla-luciferase reporter activity assay. In human T cells, each mutant Cal/0 NS1 protein was able to limit luciferase activity significantly better than the wild-type (WT) Cal/0 NS1, with the G1D mutant inhibiting approximately 0% activity (FIG. A). However, as compared with Tx/1 NS1, the block was still relatively poor. We therefore made combinations of the mutations and tested their effect on gene expression. Although luciferase inhibition with the dual RK/G1D mutant was better than WT, only the Cal/0 NS1 with all three amino-acid substitutions (RK, E1D, and G1D; TripleMut) completely blocked gene expression to a level comparable to Tx/1 NS1 (FIG. A). As confirmation, we also observed that NS1 proteins best able to block luciferase activity also limited their own expression (FIG. B). This effect was not limited to human cells, as the same overall result was obtained in swine PK-1 cells (FIGs. C and D, respectively). Downloaded from on November, 01 by guest 1

18 In order to assess whether the three RK, E1D, and G1D mutations also increased binding of Cal/0 NS1 to CPSF0, we tested the co-precipitation of in vitro transcribed and translated S-methionine labeled NS1 (WT or TripleMut) with transiently expressed FLAG-tagged CPSF0. Using high stringency conditions (00 mm NaCl), no detectable WT Cal/0 NS1 protein could be co-precipitated with FLAG-CPSF0 (FIG. A). However, the TripleMut Cal/0 NS1 clearly bound to FLAG-CPSF0 (FIG. A). The interaction of TripleMut Cal/0 NS1 with FLAG-CPSF0 was comparable to that of Tx/1 NS1 (FIG. A). We also performed immunoprecipitations using lower salt concentrations (00 mm NaCl), under which conditions a small fraction of WT Cal/0 NS1 was found to co-precipitate with FLAG-CPSF0, although binding efficiency was clearly enhanced for the TripleMut NS1 (FIG. B). The three mutations also appeared to have minimal overall effect on the ability of Cal/0 NS1 to bind synthetic dsrna, as revealed by pi:c pull-down assays (FIG. C). Together with data from the Renillaluciferase reporter activity assays, our results indicate that mutation of residues, 1, and 1 in Cal/0 NS1 to human consensus restores the ability of this NS1 protein to bind more efficiently to CPSF0 and consequently to inhibit cellular gene expression. Thus, although the amino-acid substitutions made are conservative, they clearly have a dramatic impact on a specific NS1 function. It is likely that side-chain length, as well as charge, determine overall NS1:CPSF0 complex stability. In vitro characterization of a recombinant 00 H1N1 virus expressing NS1 with the RK, E1D, and G1D amino-acid substitutions. We cloned and established a complete reverse genetics system for the Cal/0 influenza virus isolate (1). The rescued Downloaded from on November, 01 by guest 1

19 recombinant WT Cal/0 virus (rcal/0 WT) grew with identical kinetics to the parental non-recombinant Cal/0 isolate in primary differentiated HTBEs (FIG. A). We also used this reverse genetics system to generate a recombinant mutant Cal/0 virus expressing NS1 with the RK, E1D, and G1D amino-acid substitutions (TripleMut). Unavoidably, by generating the G1D amino-acid change in NS1, a VI amino-acid substitution also occurs in NEP/NS. However, this very conservative change reverts the NEP/NS residue back to human virus consensus, and would occur even if G1D arose naturally. It is unknown what effect such an amino-acid substitution has on NEP/NS, as this residue lies outwith any previously characterized functional domain. In vitro characterization of the engineered rcal/0 TripleMut virus revealed that both this and rcal/0 WT virus had similar plaque phenotypes on MDCK cells (FIG. B). Furthermore, Western blot analysis confirmed that viral protein expression in infected MDCKs was identical for both viruses (FIG. C). We also assessed replication of the rescued viruses in primary differentiated HTBEs and the swine PK-1 cell-line. Surprisingly, and in contrast to our initial hypothesis, the three NS1 mutations did not confer a selective growth advantage to rcal/0 TripMut over rcal/0 WT in the primary human cells (FIG. D), nor was this mutant significantly attenuated in the swine cell-line (FIG. E). These data also indicate that the single conservative amino-acid substitution (VI) in NS/NEP does not affect the replication of Cal/0 in vitro. We next tested the ability of the rcal/0 TripleMut virus to limit expression of IFNβ and IFN-stimulated genes during infection. Primary undifferentiated HTBEs were infected at an MOI of PFU/cell with rcal/0 WT or rcal/0 TripleMut. At various times post- Downloaded from on November, 01 by guest 1

20 infection, total RNA was extracted and relative fold-induction of the indicated viral and cellular mrnas was quantified by qpcr. As shown in FIG. A, the infectious titers reached after h by both viruses were identical. In addition, the levels of viral NA mrna increased at the same rate for both rcal/0 WT and the rcal/0 TripleMut viruses (FIG. B), consistent with the equal growth kinetics of these viruses observed in both primary differentiated HTBE cells and the swine PK-1 cell-line (FIG. ). Nevertheless, it should be noted that both replication and cytokine responses can differ depending upon the differentiation state of primary cells (). However, as expected, the amount of IFNβ mrna produced during infection with the rcal/0 TripleMut virus was significantly lower than that produced by the rcal/0 WT virus (FIG. C). Furthermore, the mrna levels of IFN-stimulated genes such as OAS, MxA, and IP (FIGs. D-F, respectively) were also significantly lower in rcal/0 TripleMut virus-infected cells, probably as a result of both their direct inhibition () and the indirect effect of IFNβ limitation. We attribute this general immune-antagonistic phenotype to the enhanced capability of the rcal/0 TripleMut NS1 protein to bind CPSF0 and block cellular gene expression. Such data are in full agreement with our observations using transiently-expressed WT and TripleMut Cal/0 NS1 proteins in the absence of virus infection (FIG. ). Furthermore, these results indicate that for WT Cal/0 virus, the viral polymerase is unable to compensate wholly for the WT NS1 deficiency (e.g. by the viral cap-snatching activity (, ), or virus-induced RNA polymerase II degradation (, )). Nevertheless, as reported for other influenza A viruses with NS1 polymorphisms that also preclude direct CPSF0 binding (, ), it may be that the cognate WT Cal/0 viral polymerase complex allows a minor fraction of WT NS1 to associate with CPSF0 during infection, Downloaded from on November, 01 by guest 0

21 thereby contributing to partial innate immune suppression at the level of pre-mrna processing. Any such inhibition is likely to be in addition to a pre-transcriptional block by NS1 at the level of RIG-I/TRIM () In vivo characterization of a recombinant 00 H1N1 virus expressing NS1 with the RK, E1D, and G1D amino-acid substitutions. To test whether increased ability of NS1 to block general gene expression would affect in vivo viral replication and/or pathogenicity, we compared the rcal/0 WT and rcal/0 TripleMut viruses in a mouse model. Initial studies using x PFU of rcal/0 WT or rcal/0 TripleMut showed that both these viruses replicated to similar average titers in the lungs of infected congenic B.AG-Mx1 mice [Day :.x PFU/mL (WT) vs..x PFU/mL (TripleMut); Day :.x PFU/mL (WT) vs..0x PFU/mL (TripleMut)]. However, no significant body weight loss after infection was observed at this dose in these mice (data not shown). DBA/J mice were therefore selected for subsequent experiments as they are reported to be highly susceptible to influenza virus infection (, 1, ). Mice were infected with PFU of each virus (or PBS alone) and body weights were monitored daily for 1 days. Surprisingly, the rcal/0 TripleMut virus caused less weight loss in infected DBA/J mice than the rcal/0 WT virus (FIG. A). Furthermore, although in the lungs of mice infected with PFU of virus the rcal/0 TripleMut appeared to reach similar mean peak infectious titers to WT [FIG. B, day : x PFU/mL (TripleMut) vs. x PFU/mL (WT)], the rcal/0 TripleMut virus seemed to be cleared slightly faster [FIG. B, day :.x PFU/mL (TripleMut) vs. x PFU/mL (WT)]. Consistent with our data in infected undifferentiated HTBEs, quantitative analysis of IFNβ mrna in infected mouse lungs indicated that the rcal/0 TripleMut virus induced Downloaded from on November, 01 by guest 1

22 less of an innate immune response than WT (FIG. C). However, we cannot exclude the possibility that this is due to slight attenuation of the TripleMut virus in vivo rather than efficient antagonism of the murine IFN system We also studied the replication abilities of the rcal/0 WT and rcal/0 TripleMut viruses in a ferret model. Ferrets were infected with TCID 0 of each virus and the amount of infectious virus present in different parts of the respiratory tract was determined at days post-infection. Consistent with the mouse data indicating that the rcal/0 TripleMut virus is cleared faster than WT, less rcal/0 TripleMut virus was detected in the nasal turbinate of ferrets at this time-point, as compared with WT (FIG. A). In addition, the amount of rcal/0 TripleMut virus in the lung appeared lower than WT (FIG. A). However, similar titers for both viruses were observed in the trachea (FIG. A). As previously described (1), the ferret model also enabled us to assess both the direct contact and aerosol respiratory droplet transmission dynamics of the two viruses. By analyzing daily nasal washes of infected and contact ferrets, we observed that total rcal/0 WT virus shedding was slightly higher than that of rcal/0 TripleMut in infected ferrets (FIGs. B and C), possibly due to faster clearance of the TripleMut virus as seen with the DBA/J mice. Nevertheless, the rcal/0 TripleMut virus still transmitted as efficiently as WT to other ferrets by both direct contact and respiratory droplet routes (FIGs. B and C). These data suggest that, in contrast to our observations in vitro, gain-of-function mutations in NS1 that enhance its ability to block general gene expression may reduce the overall replication and pathogenicity of Cal/0 in vivo. Downloaded from on November, 01 by guest

23 DISCUSSION Whilst characterizing the 00 pandemic H1N1 influenza A virus, we found that the NS1 protein of this virus lacks the ability to block general host gene expression in both human and swine cells. This is due to its inefficient binding to cellular CPSF0, a property otherwise maintained in circulating human influenza A viruses () that is reported to contribute towards suppression of host innate immune responses (, ). By identifying and making novel human-adaptive mutations in NS1, we were able to introduce this function into a recombinant prototype 00 H1N1 virus isolate, Cal/0. As with other influenza A viruses shown naturally to be defective in blocking CPSF0 function [e.g. A/Hong Kong// [HN1] ()], we found that restoration of CPSF0 binding resulted in a virus that induces less IFNβ mrna (as well as other innate immune mrnas) during infection. In order to restore efficient CPSF0 binding, we rationally mutated three residues of Cal/0 NS1 to human H1N1 virus consensus based on the recently published crystal structure of an NS1-ED in complex with CPSF0-FF (). However, other uncharacterized amino-acid changes may arise naturally that also contribute to restoration of CPSF0 binding independently of the three presented here. Nevertheless, these three amino-acids (R, E1, and G1) are highly conserved in the NS1 proteins of classically-derived swine influenza A viruses, particularly within the H1 subtype (TABLE 1). The viruses isolated from swine that have other amino-acids at these positions are either spill-over viruses from other species (only a small percentage of Downloaded from on November, 01 by guest

24 human and avian H1 influenza A viruses have the swine consensus), or swine viruses of the European avian-like lineage. Based on this conservation, and the poor ability of Sw/Tx/ NS1 to block general gene expression, we hypothesize that lack of binding and inhibition of CPSF0 is a general feature of classically-derived swine influenza A virus NS1 proteins. Indeed, the NS1 protein of the first influenza virus isolated from pigs (A/Swine/Iowa/1/10) already had R, E1, and G1. This is in stark contrast to its closest human relative, the 11 pandemic influenza virus (e.g. A/Brevig Mission/1/1), which had K, D1, and D1, and clearly binds/inhibits CPSF0 [this study, (, )]. Thus, it may be that non-binding amino-acid substitutions were selected for in the pig population after introduction of the precursor virus, and have been maintained in the classical swine NS lineage until the present day. It should be noted that such non-binding substitutions have yet to be detected in the European avian-like lineage of swine influenza A viruses that was established in 1. It is possible that complete swine adaptation has yet to occur for this new lineage, or that the classical and European avian-like lineages are evolving independently (). In this latter regard, it may be that lineage-specific functional adaptation in NS1 is generated by a different set of mutations. We are planning to test the ability of European avian-like NS1 proteins to bind/inhibit CPSF0. We had hypothesized that enhanced ability to limit IFNβ production would increase replication of the rcal/0 virus in human cells. However, we were unable to detect an increase in viral replication due to this new function, either in primary human tracheobronchial epithelial cells or in a swine cell-line. Furthermore, one might have Downloaded from on November, 01 by guest

25 expected the gain-of-function virus to grow better only in human cells, and worse in swine cells, particularly if ability of NS1 to bind CPSF0 is a human-adaptive phenotype that is selected against in pigs. Thus, it may be that our experimental set-up is not sensitive enough for detecting small phenotypic differences. It is possible that studies using additional primary substrates or cell-types (particularly of swine origin) could aid in further dissecting the effect of these NS1 adaptations. We also cannot exclude the notion that the polymorphism changes we made to the Cal/0 NS1 protein negatively affect another of the multiple NS1 functions independently of their clear positive influence on CPSF0 binding. For example, the alterations we made are at one of the proposed dimeric interfaces of the NS1 effector domain (1), which is concomitant with the CPSF0 binding interface (). Thus, slight alterations in the temporal dimeric packing of the TripleMut effector domain (or even the single amino-acid change in NEP) may have slightly reduced virus replication thereby countering any positive effect seen by restoring NS1:CPSF0 binding. Intriguingly, given that we only made naturally-occurring polymorphism changes in the NS segment, such a possibility suggests that the potential unknown function lost by these mutations may actually be important and selected for in the classical swine lineage. The contribution of NS1 to the higher replication efficiency and pathogenicity of the 00 H1N1 virus [as compared with seasonal H1N1 viruses (,, )] is unknown. Nevertheless, the NS1 protein of this pandemic virus lacks previously postulated virulence markers, such as deletion of residues 0- (), a glutamic acid at position (), or the presence of a consensus C-terminal PDZ-domain ligand motif (, ). Furthermore, experimental introduction of naturally-isolated specific functional mutations Downloaded from on November, 01 by guest

26 in the NS1 C-terminal tail did not alter replication, pathogenicity or transmission of the 00 H1N1 virus (0). In our in vivo DBA/J mouse model of infection the gain-offunction (TripleMut) virus was slightly less pathogenic, and appeared to be cleared faster, than the wild-type virus (which lacks the ability to inhibit CPSF0). Prima facie, these data suggest that inefficient CPSF0 inhibition may actually contribute to virulence of this swine-origin virus in mice. Although it has been noted that the wild-type Cal/0 virus induces high amounts of certain pro-inflammatory cytokines during in vivo infections of mice and cynomolgus macaques (), it is not known if this is due solely to a defect in NS1 function. In addition, it is unclear whether this exuberant immune response plays any significant role in disease progression. To define lack of CPSF0 binding as a bona fide virulence factor for the 00 pandemic H1N1 influenza A virus, we would have to remove this function from an unrelated influenza A virus strain that naturally inhibits CPSF0 and assess its resulting pathogenicity. It may also be that correlation of poor CPSF0 binding with virulence is a mouse-specific phenotype, particularly as many experimentally-derived mouse-adapted viruses have mutations that at least partially destabilize the interaction of NS1 with CPSF0 (, ). If this is the case, it is interesting that the TripleMut virus also demonstrated lower viral titers in the nasal turbinates of ferrets. Of note, the mouse-adapted influenza virus strain A/WSN/ (WSN/) contains asparagine at position 1, rather than the aspartic acid consensus for efficient CPSF0 binding. Thus, although WSN/ NS1 has been shown to interact with the FF domain of CPSF0 (), this may be less efficient than NS1 proteins from non-mouse adapted seasonal human viruses. If enhanced virulence in mice correlates with poor CPSF0 binding, then this may explain a previous observation whereby introduction of the BM/1 Downloaded from on November, 01 by guest

27 NS1 (which binds/inhibits CPSF0 very efficiently) into a recombinant WSN/ virus significantly reduced pathogenicity in a mouse model () The biological reasons for different influenza A viruses maintaining or not their ability to limit general gene expression via NS1 are unclear. Nevertheless, a pattern is now emerging that this may be a viral strain- and host species- specific phenotype. Our data based on residues, 1, and 1 would strongly suggest that swine influenza viruses commonly lack this function. Furthermore, analysis of residues and [two major determinants of CPSF0 binding efficiency (, )] shows that stable CPSF0 binding is also generally lost in avian H and H viruses (TABLE 1), particularly in H and H viruses that infect quail, pheasants, and partridges, rather than ducks and chickens. This observation may have already been confirmed experimentally when adapting a duck influenza virus to quail, as the resulting virus had the MT amino-acid change (). We note that a surprising proportion of 00 H1N1 viruses (approximately %) isolated since April 00 also have amino-acid substitutions at positions or in NS1 (TABLE 1). This is likely due to minimal selection pressure to maintain these CPSF0 binding residues given that the protein lacks this function already. Remarkably, most human H influenza A viruses appear to have the potential affinity-lowering glutamic acid residue at position 1, although presumably these NS1 proteins can all bind CPSF0 to some extent. Another notable subset of NS1 proteins are those classified as allele B, which are exclusively found in avian influenza A viruses [reviewed in (1)]. Almost all allele B NS1 proteins have tyrosine at position and arginine at position. Thus, there may be sliding-scale of CPSF0 binding affinities from strong to weak, Downloaded from on November, 01 by guest

28 with each particular virus (in a particular host) replicating optimally with only a certain level of CPSF0 inhibition It is still unclear whether efficient CPSF0 binding by NS1 contributes to the human adaptation of influenza A viruses. However, now that the swine-origin 00 pandemic H1N1 virus is circulating in humans, we may start to see human virus consensus emerge at residues, 1, and 1. Indeed, nine 00 pandemic H1N1 viruses with individual mutations at these positions (each with the potential to slightly increase CPSF0 binding) have already been isolated, although whether they are more fit is unknown. From a public health point of view, our in vivo data would suggest that in the current 00 H1N1 viral genetic background, such amino-acid changes in NS1 alone (or others that independently restore even a partial phenotype) will not dramatically increase virus pathogenicity, nor significantly affect virus transmissibility. Nevertheless, given our data in primary undifferentiated human tracheobronchial epithelial cells, where the rcal0 TripleMut virus seems better than WT at suppressing the host innate immune response, we cannot exclude the possibility that potential future gain of this NS1 function will enhance replication of the 00 pandemic H1N1 virus in humans. It may therefore be particularly prudent to survey newly emerging NS segment mutations, determine the corresponding NS1 protein phenotype, and assess any associations with clinical outcome. Downloaded from on November, 01 by guest

29 ACKNOWLEDGEMENTS We are grateful to Peter Palese (Mount Sinai School of Medicine, NY, USA) for helpful discussions and for critical reading of the manuscript. We thank Osman Lizardo for excellent technical assistance, and the Centers for Disease Control and Prevention (CDC Atlanta, GA, USA), Yoshihiro Kawaoka (University of Wisconsin-Madison, WI, USA), Estanislao Nistal-Villán (Mount Sinai School of Medicine, NY, USA), Randy Albrecht (Mount Sinai School of Medicine, NY, USA), Takashi Fujita (Kyoto Univeristy, Japan), Peter Staeheli (University of Freiburg, Germany), and Luis Martínez-Sobrido (University of Rochester Medical Center, NY, USA) for some of the reagents used in this study. This work was partially supported by CRIP, an NIAID funded Center for Research in Influenza Pathogenesis (contract number HHSN00000C), and by NIAID grants RO1AI, U1AI0 and PO1AI (to AG-S). JS is supported by a career development fellowship from the Northeast Biodefense Center (U AI01). ACL is a Parker B. Francis Fellow in pulmonary research. Downloaded from on November, 01 by guest

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31 Garcia-Sastre, A., A. Egorov, D. Matassov, S. Brandt, D. E. Levy, J. E. Durbin, P. Palese, and T. Muster. 1. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology : Garten, R. J., C. T. Davis, C. A. Russell, B. Shu, S. Lindstrom, A. Balish, W. M. Sessions, X. Xu, E. Skepner, V. Deyde, M. Okomo-Adhiambo, L. Gubareva, J. Barnes, C. B. Smith, S. L. Emery, M. J. Hillman, P. Rivailler, J. Smagala, M. de Graaf, D. F. Burke, R. A. Fouchier, C. Pappas, C. M. Alpuche-Aranda, H. Lopez-Gatell, H. Olivera, I. Lopez, C. A. Myers, D. Faix, P. J. Blair, C. Yu, K. M. Keene, P. D. Dotson, Jr., D. Boxrud, A. R. Sambol, S. H. Abid, K. St George, T. Bannerman, A. L. Moore, D. J. Stringer, P. Blevins, G. J. Demmler-Harrison, M. Ginsberg, P. Kriner, S. Waterman, S. Smole, H. F. Guevara, E. A. Belongia, P. A. Clark, S. T. Beatrice, R. Donis, J. Katz, L. Finelli, C. B. Bridges, M. Shaw, D. B. Jernigan, T. M. Uyeki, D. J. Smith, A. I. Klimov, and N. J. Cox. 00. Antigenic and genetic characteristics of swine-origin 00 A(H1N1) influenza viruses circulating in humans. Science : Gray, T. E., K. Guzman, C. W. Davis, L. H. Abdullah, and P. Nettesheim. 1. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 1: Guo, Z., L. M. Chen, H. Zeng, J. A. Gomez, J. Plowden, T. Fujita, J. M. Katz, R. O. Donis, and S. Sambhara. 00. NS1 protein of influenza A virus inhibits the function of intracytoplasmic pathogen sensor, RIG-I. Am J Respir Cell Mol Biol :-. 1. Hai, R., M. Schmolke, Z. T. Varga, B. Manicassamy, T. T. Wang, J. A. Belser, M. B. Pearce, A. Garcia-Sastre, T. M. Tumpey, and P. Palese. 0. PB1-F expression by the 00 pandemic H1N1 influenza virus has minimal impact on virulence in animal models. J Virol. 1. Hai, R., M. Schmolke, Z. T. Varga, B. Manicassamy, T. T. Wang, J. A. Belser, M. B. Pearce, A. Garcia-Sastre, T. M. Tumpey, and P. Palese. in press. PB1-F expression by the 00 pandemic H1N1 influenza virus has minimal impact on virulence in animal models. J Virol. 1. Hale, B. G., W. S. Barclay, R. E. Randall, and R. J. Russell. 00. Structure of an avian influenza A virus NS1 protein effector domain. Virology : Hale, B. G., R. E. Randall, J. Ortin, and D. Jackson. 00. The multifunctional NS1 protein of influenza A viruses. J Gen Virol :-. 0. Hale, B. G., J. Steel, B. Manicassamy, R. A. Medina, J. Ye, D. Hickman, A. C. Lowen, D. R. Perez, and A. Garcia-Sastre. 0. Mutations in the NS1 C- terminal tail do not enhance replication or virulence of the 00 pandemic H1N1 influenza A virus. J Gen Virol. 1. Hancock, K., V. Veguilla, X. Lu, W. Zhong, E. N. Butler, H. Sun, F. Liu, L. Dong, J. R. DeVos, P. M. Gargiullo, T. L. Brammer, N. J. Cox, T. M. Tumpey, and J. M. Katz. 00. Cross-reactive antibody responses to the 00 pandemic H1N1 influenza virus. N Engl J Med 1:1-.. Hayman, A., S. Comely, A. Lackenby, S. Murphy, J. McCauley, S. Goodbourn, and W. Barclay. 00. Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway. Virology :-. Downloaded from on November, 01 by guest 1

32 Hossain, M. J., D. Hickman, and D. R. Perez. 00. Evidence of expanded host range and mammalian-associated genetic changes in a duck HN influenza virus following adaptation in quail and chickens. PLoS One :e.. Itoh, Y., K. Shinya, M. Kiso, T. Watanabe, Y. Sakoda, M. Hatta, Y. Muramoto, D. Tamura, Y. Sakai-Tagawa, T. Noda, S. Sakabe, M. Imai, Y. Hatta, S. Watanabe, C. Li, S. Yamada, K. Fujii, S. Murakami, H. Imai, S. Kakugawa, M. Ito, R. Takano, K. Iwatsuki-Horimoto, M. Shimojima, T. Horimoto, H. Goto, K. Takahashi, A. Makino, H. Ishigaki, M. Nakayama, M. Okamatsu, D. Warshauer, P. A. Shult, R. Saito, H. Suzuki, Y. Furuta, M. Yamashita, K. Mitamura, K. Nakano, M. Nakamura, R. Brockman- Schneider, H. Mitamura, M. Yamazaki, N. Sugaya, M. Suresh, M. Ozawa, G. Neumann, J. Gern, H. Kida, K. Ogasawara, and Y. Kawaoka. 00. In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature 0:1-.. Jackson, D., M. J. Hossain, D. Hickman, D. R. Perez, and R. A. Lamb. 00. A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. Proc Natl Acad Sci U S A :1-.. Jain, S., L. Kamimoto, A. M. Bramley, A. M. Schmitz, S. R. Benoit, J. Louie, D. E. Sugerman, J. K. Druckenmiller, K. A. Ritger, R. Chugh, S. Jasuja, M. Deutscher, S. Chen, J. D. Walker, J. S. Duchin, S. Lett, S. Soliva, E. V. Wells, D. Swerdlow, T. M. Uyeki, A. E. Fiore, S. J. Olsen, A. M. Fry, C. B. Bridges, and L. Finelli. 00. Hospitalized patients with 00 H1N1 influenza in the United States, April-June 00. N Engl J Med 1:1-.. Katze, M. G., and R. M. Krug. 1. Metabolism and expression of RNA polymerase II transcripts in influenza virus-infected cells. Mol Cell Biol :1-0.. Kochs, G., A. Garcia-Sastre, and L. Martinez-Sobrido. 00. Multiple antiinterferon actions of the influenza A virus NS1 protein. J Virol 1:0-1.. Kuo, R. L., and R. M. Krug. 00. Influenza a virus polymerase is an integral component of the CPSF0-NS1A protein complex in infected cells. J Virol : Lange, E., D. Kalthoff, U. Blohm, J. P. Teifke, A. Breithaupt, C. Maresch, E. Starick, S. Fereidouni, B. Hoffmann, T. C. Mettenleiter, M. Beer, and T. W. Vahlenkamp. 00. Pathogenesis and transmission of the novel swine-origin influenza virus A/H1N1 after experimental infection of pigs. J Gen Virol 0:-. 1. Li, Y., Z. Y. Chen, W. Wang, C. C. Baker, and R. M. Krug The '-endprocessing factor CPSF is required for the splicing of single-intron pre-mrnas in vivo. RNA :0-1.. Livak, K. J., and T. D. Schmittgen Analysis of relative gene expression data using real-time quantitative PCR and the (-Delta Delta C(T)) Method. Methods :0-.. Long, J. X., D. X. Peng, Y. L. Liu, Y. T. Wu, and X. F. Liu. 00. Virulence of HN1 avian influenza virus enhanced by a 1-nucleotide deletion in the viral nonstructural gene. Virus Genes :1-.. Maines, T. R., A. Jayaraman, J. A. Belser, D. A. Wadford, C. Pappas, H. Zeng, K. M. Gustin, M. B. Pearce, K. Viswanathan, Z. H. Shriver, R. Raman, Downloaded from on November, 01 by guest

33 N. J. Cox, R. Sasisekharan, J. M. Katz, and T. M. Tumpey. 00. Transmission and pathogenesis of swine-origin 00 A(H1N1) influenza viruses in ferrets and mice. Science :-.. Manicassamy, B., R. A. Medina, R. Hai, T. Tsibane, S. Stertz, E. Nistal- Villan, P. Palese, C. F. Basler, and A. Garcia-Sastre. 0. Protection of mice against lethal challenge with 00 H1N1 influenza A virus by 11-like and classical swine H1N1 based vaccines. PLoS Pathog :e00.. Mibayashi, M., L. Martinez-Sobrido, Y. M. Loo, W. B. Cardenas, M. Gale, Jr., and A. Garcia-Sastre. 00. Inhibition of retinoic acid-inducible gene I- mediated induction of beta interferon by the NS1 protein of influenza A virus. J Virol 1:1-.. Moss, B., O. Elroy-Stein, T. Mizukami, W. A. Alexander, and T. R. Fuerst. 10. Product review. New mammalian expression vectors. Nature :1-.. Munster, V. J., E. de Wit, J. M. van den Brand, S. Herfst, E. J. Schrauwen, T. M. Bestebroer, D. van de Vijver, C. A. Boucher, M. Koopmans, G. F. Rimmelzwaan, T. Kuiken, A. D. Osterhaus, and R. A. Fouchier. 00. Pathogenesis and transmission of swine-origin 00 A(H1N1) influenza virus in ferrets. Science :1-.. Nemeroff, M. E., S. M. Barabino, Y. Li, W. Keller, and R. M. Krug. 1. Influenza virus NS1 protein interacts with the cellular 0 kda subunit of CPSF and inhibits 'end formation of cellular pre-mrnas. Mol Cell 1: Newby, C. M., L. Sabin, and A. Pekosz. 00. The RNA binding domain of influenza A virus NS1 protein affects secretion of tumor necrosis factor alpha, interleukin-, and interferon in primary murine tracheal epithelial cells. J Virol 1: Niwa, H., K. Yamamura, and J. Miyazaki. 11. Efficient selection for highexpression transfectants with a novel eukaryotic vector. Gene :1-.. Noah, D. L., K. Y. Twu, and R. M. Krug. 00. Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the ' end processing of cellular pre-mrnas. Virology 0:-.. Obenauer, J. C., J. Denson, P. K. Mehta, X. Su, S. Mukatira, D. B. Finkelstein, X. Xu, J. Wang, J. Ma, Y. Fan, K. M. Rakestraw, R. G. Webster, E. Hoffmann, S. Krauss, J. Zheng, Z. Zhang, and C. W. Naeve. 00. Largescale sequence analysis of avian influenza isolates. Science :1-0.. Opitz, B., A. Rejaibi, B. Dauber, J. Eckhard, M. Vinzing, B. Schmeck, S. Hippenstiel, N. Suttorp, and T. Wolff. 00. IFNbeta induction by influenza A virus is mediated by RIG-I which is regulated by the viral NS1 protein. Cell Microbiol :0-.. Pichlmair, A., O. Schulz, C. P. Tan, T. I. Naslund, P. Liljestrom, F. Weber, and C. Reis e Sousa. 00. RIG-I-mediated antiviral responses to single-stranded RNA bearing '-phosphates. Science 1:-01.. Plotch, S. J., M. Bouloy, I. Ulmanen, and R. M. Krug. 11. A unique cap(mgpppxm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell :-. Downloaded from on November, 01 by guest

34 Quinlivan, M., D. Zamarin, A. Garcia-Sastre, A. Cullinane, T. Chambers, and P. Palese. 00. Attenuation of equine influenza viruses through truncations of the NS1 protein. J Virol :1-.. Rodriguez, A., A. Perez-Gonzalez, and A. Nieto. 00. Influenza virus infection causes specific degradation of the largest subunit of cellular RNA polymerase II. J Virol 1:1-.. Seo, S. H., E. Hoffmann, and R. G. Webster. 00. Lethal HN1 influenza viruses escape host anti-viral cytokine responses. Nat Med : Solorzano, A., R. J. Webby, K. M. Lager, B. H. Janke, A. Garcia-Sastre, and J. A. Richt. 00. Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs. J Virol :-. 1. Sorrell, E. M., H. Wan, Y. Araya, H. Song, and D. R. Perez. 00. Minimal molecular constraints for respiratory droplet transmission of an avian-human HN influenza A virus. Proc Natl Acad Sci U S A :-0.. Srivastava, B., P. Blazejewska, M. Hessmann, D. Bruder, R. Geffers, S. Mauel, A. D. Gruber, and K. Schughart. 00. Host genetic background strongly influences the response to influenza a virus infections. PLoS One :e.. Staeheli, P., P. Dreiding, O. Haller, and J. Lindenmann. 1. Polyclonal and monoclonal antibodies to the interferon-inducible protein Mx of influenza virusresistant mice. J Biol Chem 0:-.. Swayne, D. E., M. Pantin-Jackwood, D. Kapczynski, E. Spackman, and D. L. Suarez. 00. Susceptibility of poultry to pandemic (H1N1) 00 Virus. Emerg Infect Dis 1:01-.. Talon, J., C. M. Horvath, R. Polley, C. F. Basler, T. Muster, P. Palese, and A. Garcia-Sastre Activation of interferon regulatory factor is inhibited by the influenza A virus NS1 protein. J Virol :-.. Twu, K. Y., R. L. Kuo, J. Marklund, and R. M. Krug. 00. The HN1 influenza virus NS genes selected after 1 enhance virus replication in mammalian cells. J Virol 1:-1.. Twu, K. Y., D. L. Noah, P. Rao, R. L. Kuo, and R. M. Krug. 00. The CPSF0 binding site on the NS1A protein of influenza A virus is a potential antiviral target. J Virol 0:-.. Vreede, F. T., A. Y. Chan, J. Sharps, and E. Fodor. 00. Mechanisms and functional implications of the degradation of host RNA polymerase II in influenza virus infected cells. Virology.. Wang, X., M. Li, H. Zheng, T. Muster, P. Palese, A. A. Beg, and A. Garcia- Sastre Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon. J Virol :-. 0. Yoneyama, M., W. Suhara, Y. Fukuhara, M. Fukuda, E. Nishida, and T. Fujita. 1. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF- and CBP/p00. EMBO J 1:-. Downloaded from on November, 01 by guest

35 FIGURE LEGENDS FIGURE 1. Inhibition of IFNβ induction by different influenza A virus NS1 proteins. T cells were co-transfected for 1 h with a pcaggs expression plasmid encoding the indicated NS1 protein (or GST) together with a FF-luciferase IFNβpromoter reporter plasmid (p1luc). After infection with SeV for a further 1 h luciferase (FF-Luc) activity was determined. Zoomed inset highlights differences between NS1 proteins. Results represent the means and standard deviations of triplicate values (normalized to GST + SeV) obtained in a single experiment, and are representative of two independent experiments. FIGURE. The Cal/0 NS1 protein is unable to block general gene expression in human or swine cells. Human T (A), or swine PK-1 (C) cells were co-transfected with a pcaggs expression plasmid encoding the indicated NS1 protein (or GST) together with a constitutively active Renilla-luciferase plasmid. Luciferase activity was determined h post-transfection. Results show the means and standard deviations of triplicate values [normalized to GST (A), or PR/ NS1 (C)] obtained in a single experiment, and are representative of two independent experiments. (B) Western blot analysis of lysates from (A). (D) Western blot analysis of lysates from (C). NS1 and GST were detected using a polyclonal rabbit anti-serum raised against a fusion protein of GST and the N-terminal RNA binding domain of NS1. Tubulin acted as a loading control. Molecular weight markers (kda) are indicated to the right. Downloaded from on November, 01 by guest

36 FIGURE. Residues at the interface of the NS1:CPSF0 complex. (A) Amino-acid sequence alignment of the NS1 effector domains (ED, residues -0) from PR/, BM/1, Tx/1, Sw/Tx/, and Cal/0. Identity (*) and level of similarity (: or.) are indicated. Gray shading highlights residues of NS1 previously shown experimentally to be important for CPSF0 binding. Black boxes highlight residues common to PR/, BM/1, and Tx/1 NS1-EDs ( human-like ), but different in Sw/Tx/ and Cal/0 NS1- EDs ( swine-like ). Residues, 1, and 1 (which are the only residues unique to Sw/Tx/ and Cal/0 NS1 proteins at the NS1:CPSF0 interface, see below) are labeled. (B) Structural representation of NS1 in complex with the FF fragment of CPSF0. Monomeric NS1-ED is shown in cartoon format (green), whilst the CPSF0-FF fragment is shown in surface representation (blue). Unique residues of Sw/Tx/ and Cal/0 NS1-EDs compared to PR/, BM/1, and Tx/1 are highlighted with black spheres. (C) Close-up view of the NS1:CPSF0 interface with residues different in Sw/Tx/ and Cal/0 NS1-EDs highlighted in black (sticks). Panels B and C were generated using MacPyMol [PDB ID: RHK ()]. FIGURE. Three amino-acid substitutions restore ability of the Cal/0 NS1 protein to block general gene expression in human and swine cells. Human T (A), or swine PK-1 (C) cells were co-transfected with a pcaggs expression plasmid encoding the indicated WT NS1 protein or the Cal/0 mutants: RK, E1D, G1D, RK/G1D (/1), or RK/E1D/G1D (TripleMut); together with a constitutively active Renilla-luciferase plasmid. h post-transfection luciferase activity was determined. Results show the means and standard deviations of triplicate values (normalized to WT Cal/0 NS1) obtained in a single experiment, and are representative Downloaded from on November, 01 by guest

37 of two independent experiments. (B and D) Western blot analysis of lysates from (A) and (C), respectively. NS1 and tubulin were detected as for FIG FIGURE. Interaction of CPSF0 with WT Cal/0 NS1 or TripleMut Cal/0 NS1. FLAG-tagged CPSF0 was expressed in T cells (CPSF0), mixed with in vitro synthesized S-methionine labeled NS1 (WT Cal/0, TripleMut Cal/0, or HA-tagged Tx/1), and immunoprecipitated using anti-flag resin. T lysates without FLAGtagged CPSF0 (Mock) acted as a negative control. Following SDS-PAGE, precipitated proteins were detected by Coomassie blue staining (IgG), Western blot (FLAG-tagged CPSF0), or phosphorimaging ( S-methionine labeled NS1). Immunoprecipitations were performed using 00 mm NaCl (A, In denotes % input) or 00 mm NaCl (B, In denotes 1% input). (C) Ability of WT Cal/0 NS1 or TripleMut Cal/0 NS1 to bind synthetic dsrna. Lysates from A cells infected with rcal/0 WT or rcal/0 TripleMut (MOI of PFU/cell) were precipitated with pi:c-sepharose (pi:c) or Sepharose only (-). Following SDS-PAGE, NS1 proteins were detected by Western blot. Molecular weight markers (kda) are indicated to the right. FIGURE. In vitro characterization of the rcal/0 TripleMut virus. (A) Multicycle growth analysis of rcal/0 WT and non-recombinant Cal/0 WT viruses in primary differentiated human airway epithelial cells. (B) Plaque phenotype of rcal/0 WT and rcal/0 TripleMut viruses in MDCK cells. (C) Western blot determination of viral NP and NS1 protein expression in MDCK cells infected for 1 h with the rcal/0 WT or rcal/0 TripleMut (TM) viruses (MOI of PFU/cell). Molecular weight markers (kda) are indicated to the right. Multicycle growth analysis of rcal/0 WT and rcal/0 Downloaded from on November, 01 by guest

38 TripleMut viruses in primary differentiated human airway epithelial cells (D), and the swine PK-1 cell-line (E). Data points for growth curves show mean values (n=) and error bars represent standard deviation FIGURE. Ability of rcal/0 TripleMut virus to limit expression of IFNβ and IFNstimulated genes during infection. Primary undifferentiated human airway epithelial cells were infected at an MOI of PFU/cell with rcal/0 WT or rcal/0 TripleMut viruses. (A) Infectious titers determined by plaque assay in supernatants after h. (B-F) Cells were lysed at the times indicated and total RNA extracted. Following reverse transcription using oligo(dt) the levels of viral NA mrna (B), IFNβ mrna (C), OAS mrna (D), MxA mrna (E), and IP mrna (F) were quantified in triplicate by qpcr. Values were averaged and normalized to 1S RNA. Mean fold-induction relative to mrna levels in mock-infected cells is shown. Error bars represent standard deviation. Means and standard deviations were calculated from biological triplicates. FIGURE. Replication and pathogenicity of the rcal/0 TripleMut virus in mice. - week old DBA/J mice were infected intranasally with PFU of rcal/0 WT or rcal/0 TripleMut viruses. PBS-treated mice acted as a negative control. (A) Body weights were determined daily. Data show mean body weight (n=). Error bars represent standard deviations. Significance was determined using a two-tailed Student s t-test (**, p < 0.01, *, p < 0.0). (B) Lung titers were determined on days,, and post-infection from three DBA/J mice infected in parallel with PFU of rcal/0 WT or rcal/0 Downloaded from on November, 01 by guest

39 TripleMut viruses. Bars represent mean values. (C) Murine IFNβ mrna was quantified from the lung homogenates used in (B) by qpcr as for FIG FIGURE. Replication and transmission dynamics of the rcal/0 TripleMut virus in ferrets. - month old female Fitch ferrets were infected intranasally with TCID 0 of rcal/0 WT or rcal/0 TripleMut viruses. (A) Viral titers in nasal turbinates, trachea and lungs were determined on day post-infection from two infected ferrets. Bars represent mean values. (B-C) Nasal wash titers for rcal/0 WT (B) and rcal/0 TripleMut (C) viruses were determined from infected ferrets (n=), direct contact ferrets (n=), and aerosol (large and small respiratory droplet) contact ferrets (n=) on the days indicated. Bars (dark and light gray) represent the raw results of two independent experiments. To limit the number of animals used, the rcal/0 WT-infected ferret data shown are the same as previously described (0). All experiments were performed in parallel to allow fair comparison. Downloaded from on November, 01 by guest

40 TABLE 1. Species- and subtype- specific identity of NS1 residues involved in CPSF0 binding. % of NS1 sequences with specific residues at stated positions a,b 1 1 F c Y X d M c X d K c R X D d c Avian e H H H H Human e H H H1N Swine e H1 0 1 H a Percentage of NS1 sequences with certain amino-acid residues located at positions,,, 1, and 1. b All sequences are derived from publicly-available databases (as of November 00, or April 0 for the 00 H1N1 virus) and were aligned prior to analysis. c Bold, underlined residues contribute to stable CPSF0 binding. d X represents other amino-acids. e The number of sequences for each group was as follows: Avian E X d D c G X d Downloaded from on November, 01 by guest 0

41 (H1=, H=, H=00, H=), Human (H1=, H=1, 00 H1N1=1), and Swine (H1=0, H=). Values are rounded to the nearest whole percentage point. Downloaded from on November, 01 by guest 1

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