Immunopathogenic and Antibacterial Effects of H3N2 Influenza A Virus PB1-F2 Map to Amino Acid Residues 62, 75, 79, and 82

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1 JOURNAL OF VIROLOGY, Dec. 2011, p Vol. 85, No X/11/$12.00 doi: /jvi Copyright 2011, American Society for Microbiology. All Rights Reserved. Immunopathogenic and Antibacterial Effects of H3N2 Influenza A Virus PB1-F2 Map to Amino Acid Residues 62, 75, 79, and 82 Irina V. Alymova, 1 Amanda M. Green, 1 Nicholas van de Velde, 1 Julie L. McAuley, 2 Kelli L. Boyd, 3 Hazem E. Ghoneim, 1 and Jonathan A. McCullers 1 * Department of Infectious Diseases, St. Jude Children s Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee ; Department of Immunology and Microbiology, University of Melbourne, 801 Swanston St., Victoria 3053, Australia 2 ; and Department of Pathology, Division of Comparative Medicine, Vanderbilt University, 2201 West End Ave., Nashville, Tennessee Received 2 August 2011/Accepted 13 September 2011 The influenza A virus protein PB1-F2 has been linked to the pathogenesis of both primary viral and secondary bacterial infections. H3N2 viruses have historically expressed full-length PB1-F2 proteins with either proinflammatory (e.g., from influenza A/Hong Kong/1/1968 virus) or noninflammatory (e.g., from influenza A/Wuhan/359/1995 virus) properties. Using synthetic peptides derived from the active C-terminal portion of the PB1-F2 protein from those two viruses, we mapped the proinflammatory domain to amino acid residues L62, R75, R79, and L82 and then determined the role of that domain in H3N2 influenza virus pathogenicity. PB1-F2-derived peptides containing that proinflammatory motif caused significant morbidity, mortality, and pulmonary inflammation in mice, manifesting as increased acute lung injury and the presence of proinflammatory cytokines and inflammatory cells in the lungs compared to peptides lacking this motif, and better supported bacterial infection with Streptococcus pneumoniae. Infections of mice with an otherwise isogenic virus engineered to contain this proinflammatory sequence in PB1-F2 demonstrated increased morbidity resulting from primary viral infections and enhanced development of secondary bacterial pneumonia. The presence of the PB1-F2 noninflammatory (P62, H75, Q79, and S82) sequence in the wild-type virus mediated an antibacterial effect. These data suggest that loss of the inflammatory PB1-F2 phenotype that supports bacterial superinfection during adaptation of H3N2 viruses to humans, coupled with acquisition of antibacterial activity, contributes to the relatively diminished frequency of severe infections seen with seasonal H3N2 influenza viruses in recent decades compared to their first 2 decades of circulation. Downloaded from Influenza A viruses (IAVs) of the H3N2 subtype have been an important public health threat since the pandemic of 1968 (16). The pandemic strain was a reassortant, composed of 6 gene segments from the previously circulating H2N2 viruses but with hemagglutinin (HA) and polymerase basic protein 1 (PB1) segments derived from an avian source (15). It infected about 40% of the human population, causing more than 1 million deaths worldwide (8). Since that time, H3N2 influenza viruses have caused severe seasonal influenza on average every two to three influenza seasons. However, although regional differences exist, influenza-like illness attributed to H3N2 IAV strains has generally declined over the decades, and the virus appears to have adapted to humans during that time period (9). In parallel, a decline in attributable all-cause and excess mortality has also been seen (8, 23). The mechanisms by which H3N2 viruses become more or less virulent in humans are not clear. IAVs contain an eight-segment, negative-strand RNA genome. In addition to PB1, segment 2 of most IAV strains encodes a small (up to 90-amino-acid [aa]) accessory PB1-F2 protein in the 1 open reading frame (ORF) (3). A third product, N40, can also be produced from an upstream start site * Corresponding author. Mailing address: Department of Infectious Diseases, St. Jude Children s Research Hospital, 262 Danny Thomas Place, Memphis, TN Phone: (901) Fax: (901) jon.mccullers@stjude.org. Published ahead of print on 21 September in the PB1 ORF (26). Since its discovery in 2001 (3), PB1-F2 has been studied as a potentially important viral virulence factor because of a link to the pathogenicity of strains such as the highly pathogenic avian influenza viruses of the H5N1 subtype and the 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2) pandemic influenza viruses (7, 10, 18, 19). Several mechanisms for the activity of PB1-F2 have been proposed. Certain laboratory-adapted strains can cause apoptotic cell death in vitro through a mechanism dependent on interactions with mitochondrial proteins, but this function may not be generalizable (3, 18, 27). PB1-F2 expression may also alter polymerase activity or replication kinetics in vitro, although the effects are cell type and virus specific and have not yet been shown to translate into changes in pathogenesis (17, 20). The in vivo effects of PB1-F2 appear to be largely mediated through interactions of this accessory protein with the immune system, either through potentiation of inflammatory responses (18, 19) or blockade of early type I interferon pathways (6). The ability of IAVs to generate excessive inflammation in the lungs appears to be a hallmark of virulent pandemic strains (14). In vitro and in mouse models, the PB1-F2 protein plays an important role in the development of IAV-related immunopathological consequences. We recently demonstrated that PB1-F2 proteins from the pandemic influenza viruses of 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2) and from highly pathogenic H5N1 influenza virus strains of avian origin enhance virus-mediated lung inflammation via an elevation of cytokine levels in bronchoalveolar lavage (BAL) fluid and a on November 7, 2018 by guest 12324

2 VOL. 85, 2011 ROLE OF H3N2 PB1-F2 IN INFLUENZA PATHOGENESIS FIG. 1. Amino acid sequences of H3N2 lineage PB1-F2 proteins. Sequences are pictured for selected H3N2 strains that are representative of the evolution of this protein. Black shading indicates identity within this set of strains; gray and white highlight differences, dark blue shading highlights amino acids determined to be critical for the proinflammatory phenotype, and light blue shading highlights amino acids that differ between the pandemic strain HK68 and the seasonal strain Wuh95 but that were not necessary for the phenotype. Udo72, A/Udorn/307/72; Mem74, A/Memphis/101/74; Alb78, A/Albany/14/78; HK87, A/Hong Kong/7/87; Pan99, A/Panama/2007/99; NY10, A/New York/20343/10. significant influx of neutrophils and macrophages into the lungs (18). These enhanced inflammatory responses contributed to morbidity and mortality during primary viral infections when the 1918 or H5N1 influenza virus PB1-F2 proteins were present (reference 19 and unpublished data). However, this ability was lost during adaptation into seasonal strains either through truncation (in the H1N1 lineage) or mutation (in the H3N2 lineage) (18). In addition, PB1-F2 proteins from 1918 or 1934 H1N1 influenza viruses were capable of greatly enhancing secondary bacterial infections, manifesting as increased bacterial titers, more frequent progression to pneumonia, and elevated mortality rates. We speculated that the proinflammatory features of PB1-F2 must be important for maintenance of influenza viruses in the avian reservoir but that they are either neutral or evolutionarily detrimental to the virus when expressed in the mammalian lung. In the current study, we took advantage of the disparate responses noted with PB1-F2 proteins from two H3N2 influenza viruses, the 1968 pandemic strain A/Hong Kong/1/68 (here referred to as HK68) and a recent seasonal strain, A/Wuhan/359/95 (here referred to as Wuh95) (18). While the HK68 pandemic PB1-F2 protein generated strong inflammatory responses in vitro when given alone as a C-terminal peptide, the PB1-F2 protein from the Wuh95 strain, which is descended from strain HK68, did not. Neither PB1-F2 protein was capable of inducing apoptosis or altering viral replication (18, 20). Here we present an analysis of the contribution of the specific amino acids that are required for the proinflammatory phenotype in an H3N2 background, using sequence differences in the two virus strains as a guide. We demonstrate that changes at four positions in the C terminus of PB1-F2 are sufficient to confer proinflammatory activity on Wuh95 PB1-F2 or to abrogate such activity in HK68. Surprisingly, the noninflammatory Wuh95 PB1-F2 protein is shown here to possess antibacterial activity mediated by those specific amino acids. This function may make a further, unexpected contribution to the attenuation of this strain, relative to its more pathogenic pandemic forbear, through suppression of secondary bacterial pneumonia. MATERIALS AND METHODS Peptides. By the use of the predicted amino acid sequences of the HK68 and Wuh95 PB1-F2 proteins (Fig. 1), peptides from the C-terminal ends (aa 61 through 87) with no, single, or multiple substitutions at positions 62, 73, 75, 79, and 82 (following the PB1-F2 amino acid numbering) were synthesized by Gen- Script Corp. (Piscataway, NJ). Mutations to the Wuh95 PB1-F2 peptide (Pro62 to Leu [P62L]; Arg73 to Lys [R73K]; His75 to Arg [H75R]; Gln79 to Arg [Q79R]; and Ser82 to Leu [S82L]) were made to emulate the proposed proinflammatory residues found naturally in HK68. A peptide with the four key P62L, H75R, Q79R, and S82L mutations was designated Wuh95 4. Similarly, peptides were made on a HK68 background with reciprocal mutations (Leu62 to Pro [L62P]; Lys73 to Arg [K73R]; Arg75 to His [R75H]; Arg79 to Gln [R79Q]; and Leu82 to Ser [L82S]). A peptide with the inverse four mutations, L62P, R75H, R79Q, and L82S, in the HK68 background was designated HK68-4. Peptides, provided as a lyophilized powder, were solubilized in cell culture media or phosphate-buffered saline (PBS; ph 7.2) before use in in vitro or in vivo experiments, respectively. Cell cultures. MDCK (Madin-Darby canine kidney) epithelium cells were grown in 1 minimum essential medium (MEM) that contained 5% fetal bovine serum. 293T (human kidney) epithelium cells and J774 (mouse macrophage) cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Infectious agents. A chimeric influenza virus containing the hemagglutinin (HA) and NA gene segments of influenza virus A/Puerto Rico/8/34 (H1N1; here referred to as PR8) and the six internal gene segments of Wuh95 (here referred to as Wuh95/PR8) was generated by reverse genetics as previously described (12). Before rescue, the PB1 gene segment of Wuh95 was modified using sitedirected mutagenesis (QuikChange; Stratagene, La Jolla, CA) by previously described methods (19) to generate a second virus variant possessing P62L, H75R, and S82L mutations in the PB1-F2 ORF (here referred to as Wuh95 3/PR8). A mutant with the Q79R change could not be rescued, likely because it would have caused a nonsynonymous change in the PB1 ORF. The rescued chimeric viruses were amplified once in MDCK cells for stocks, and the PB1 gene segments were fully sequenced to confirm that they were free of mutations other than those described. Infectivity of the rescued viruses was determined using plaque assays in MDCK cells, as described elsewhere (5). Streptococcus pneumoniae strain A66.1 (type 3) was grown in Todd-Hewitt broth (Difco Laboratories, Detroit, MI) to an optical density at 620 nm (OD 620 ) of approximately 0.4 and then frozen at 80 C after being mixed 2:1 with 5% sterile glycerol. The titers of the frozen stocks were quantitated on tryptic soy agar (Difco Laboratories, Detroit, MI) supplemented with 3% (vol/vol) sheep erythrocytes. Cell culture assays. To determine cytokine concentrations, J774 cells in 6-well plates were treated with a panel of H3N2 PB1-F2 peptides at doses ranging from 100 to 500 M in DMEM containing 3% bovine serum albumin. Culture supernatant fluids were collected 4 h after treatment and frozen before use. Bacterial culture assays. To determine the inhibition of S. pneumoniae growth induced by H3N2 PB1-F2 peptides, the bacterial suspension was adjusted to CFU/ml and treated with PBS or peptides dissolved in Todd-Hewitt broth (Difco Laboratories, Detroit, MI) at final concentrations of 1, 10, 100, and 500 M for2or6hat37 C. The bactericidal effect was determined by plating serial dilutions of the bacterial suspension onto Todd-Hewitt agar plates supplemented with 3% sheep red blood cells (RBCs), followed by 20 h of incubation at 37 C. Mice. Experiments using 8-week-old female BALB/c or DBA/2 mice (Jackson Laboratories, Bar Harbor, ME) were performed in a biosafety level 2 facility in the Animal Resources Center at St. Jude Children s Research Hospital (SJCRH). Animals were given general anesthesia that consisted of 2.5% inhaled isoflurane (Baxter Healthcare Corporation, Deerfield, IL) prior to all interventions, and all studies were approved by the Animal Care and Use Committee at SJCRH.

3 12326 ALYMOVA ET AL. J. VIROL. Peptide studies with mice. All studies involving peptides were performed using BALB/c mice. To determine the pathogenicity of H3N2 PB1-F2 peptides, mice (10 per group) were given 60 mg of Wuh95, Wuh95 4, HK68, or HK68-4 PB1-F2 peptides once, in a intranasally (i.n.) administered volume of 100 l, and monitored for weight loss and survival. For lung pathology experiments, mice (3 per group) were administered 40 mg of PB1-F2 peptides or were administered PBS and were euthanized 3 days later. To analyze the cellular immune response and levels of cytokines, the BAL fluids from at least 5 mice per group were collected at day 3 after administration of 40 mg of H3N2 PB1-F2 peptides or PBS. In a bacterial pneumonia model, mice were exposed to 40 mg of peptides and challenged 1 day later with 2,000 CFU of S. pneumoniae per mouse. Control exposed animals received PBS instead of peptide. Weight changes (calculated for each mouse as a percentage of its weight on day 0 before peptide administration) and survival of mice were monitored for 21 days after the bacterial challenge. For bacterial titer calculations, lungs of 3 to 5 mice per group were collected on days 1, 3, and 5 after bacterial challenge. Histopathologic analysis. Lungs were removed immediately following euthanasia, sufflated, and fixed in 10% neutral buffered formalin overnight. The lungs were processed routinely, embedded in paraffin, cut into 5- m sections, stained with hematoxylin and eosin, and examined microscopically for histopathologic alterations. The lungs were assigned a grade 0 to 3 based on the histologic character of the lesions. A score of 0 was given when no pathological changes could be detected. A score of 1 was given to findings representing mild pathologies, including minimal infiltrates of lymphocytes and plasma cells around airways and vessels, minimal epithelial hyperplasia, minimal leukocyte infiltration of alveolar spaces, and 10% of the lung affected. A score of 2 was given for findings representing moderate pathologies, including moderate infiltrates of lymphocytes and plasma cells around airways and vessels, moderate epithelial hyperplasia with focal necrosis, focally extensive infiltration of the alveolar spaces by leukocytes with some consolidation, focal pleuritis, and 10% but 30% of the lung affected. A score of 3 was given for findings representing pathologies of greater severity, including extensive necrosis of airway epithelium and of interstitium, extensive leukocyte infiltration and consolidation, severe pleuritis, and lobar involvement. Grading and descriptions of pathology were performed by an experienced veterinary pathologist (K.L.B.) in a single-blinded manner with respect to the purpose of the study and the composition of the groups. Virus studies with mice. All studies involving chimeric Wuh95/PR8 and Wuh95 3/PR8 influenza viruses were performed using DBA/2 mice. To determine the pathogenicity of Wuh95/PR8 and Wuh95 3/PR8, mice (10 per group) were infected i.n. with viruses at doses ranging from 10 4 to 10 6 PFU per mouse in 100 l of sterile PBS and were monitored for 21 days for weight loss, length of survival, and death. To compare results with respect to growth of chimeric viruses, histopathologic changes in the lungs, and cellular immune response and the presence of cytokines in the BAL fluids, mice were infected i.n. with a dose of PFU per mouse. At 6, 12, and 18 h and at 1, 3, 5, 7, 9, or 11 days after infection, lungs from three or more mice from each group were harvested for virus titers. Histopathologic changes in the mouse lungs (from 3 mice per group) were determined at day 3 and 7 postinfection (p.i.). The cellular immune response in the BAL fluids from at least 5 mice per group was analyzed at 3 and 9 days p.i. In a secondary bacterial pneumonia model, mice were infected with PFU of Wuh95/PR8 and Wuh95 3/PR8 per mouse and challenged 7 days later with 100 CFU of S. pneumoniae per mouse. Control infected animals received PBS initially instead of virus. Weight changes (calculated for each mouse as a percentage of its weight on day 0 before virus infection) of mice were monitored for 21 days after the bacterial challenge. Viral and bacterial titers in the mouse lungs (at least 5 per group) as well as the cellular immune response and levels of cytokines in the BAL fluids were determined at day 2 after bacterial challenge. Lung viral and bacterial titers. The lungs were removed under sterile conditions, washed three times with PBS, homogenized, and suspended in PBS (total volume, 1 ml). The suspensions for virus titration were centrifuged at 2,000 g for 10 min to clear cellular debris. Virus titers were determined using plaque assays with MDCK cells. Lung homogenates were used directly for bacterial cultures prior to centrifugation. Pneumococcal colony counts were done by using 10-fold dilutions on tryptic soy agar plates (Difco, Laboratories, Detroit, MI) supplemented with 3% (vol/vol) sheep erythrocytes and kanamycin (400 g/ml). Analysis of cellular immune response in BAL fluid. Following euthanasia by CO 2 inhalation, the trachea was exposed and cannulated with a 24-gauge plastic catheter (Becton Dickinson Infusion Therapy Systems, Inc., Sandy, UT). Lungs were subjected to lavage twice with 1.5 ml of cold, sterile Hank s buffered salt solution supplemented with 0.05 mm EDTA. The number of white blood cells (WBCs) per milliliter of the resulting suspension was then determined using a Hemavet 3700 hematology analyzer (Drew Scientific, Dallas, TX) and a 100- l aliquot. Flow cytometry (LSRII; Becton Dickinson, San Jose, CA) was performed on the BAL fluid suspension after incubation of cells with 75 l of a 1:200 dilution of Fc blocking solution (anti-mouse CD16/CD32; BD Bioscience Inc., San Jose, CA) on ice via staining (1 l/10 6 cells) with the following anti-mouse antibodies: CD11c (efluor 450), F4-80 (fluorescein isothiocyanate [FITC]), major histocompatibility complex class II (MHC-II) (phycoerythrin [PE]), Ly6G (PerCp-Cy5.5), Ly6C (allophycocyanin [APC]), and CD11b (APC-eFluor 780) (ebioscience Inc., San Diego, CA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR): viable cells were gated from forward-scatter (FSC) and side-scatter (SSC) plots, and then different inflammatory cell subsets were gated using the following markers: for neutrophils, CD11b hi, CD11c lo F4-80 lo, and Ly6G hi Ly6C int ; for exudate macrophages, CD11b hi, CD11c lo-int F4-80 lo-int, and Ly6C hi ly6g int ; and for inflammatory dendritic cells, CD11b hi, Ly6G lo-int, and CD11c int-hi MHC-II hi. Analysis of BAL fluid cell composition was performed on the basis of the proportion of viable events analyzed by flow cytometry related to the number of WBCs per milliter. Analysis of cytokines and chemokines. Cytokines were analyzed in culture supernatants from J774 cells and BAL fluids of mice. Before measurement, BAL fluids were centrifuged at 10,000 g for 5 min and the supernatants were frozen. The concentrations of interleukin-6 (IL-6) and chemokines (KC), granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1 ), and tumor necrosis factor alpha (TNF- ) were measured by using the mouse 22-plex cytokine assay (Millipore Co., Billerica, MA) and read using a Luminex xponent 3.1 system (Bio-Rad Laboratories, Austin, TX) according to the manufacturer s recommended procedure. Samples were diluted 1:2 and 1:4 and processed in triplicate experiments in all assays with appropriate internal controls. TNF- was selected for display in the figures, as it has been shown to be broadly representative of that group of proinflammatory cytokines in this model (24). Statistical analyses. Comparison of survival data from groups of mice was done by using the log-rank chi-square test to analyze the Kaplan-Meier survival data over the period of 21 days. The mean number of days until death was estimated as the number of days that the mice survived after peptide administration or viral or bacterial infection. If no death occurred during the observation period, the mean number of days to death was considered to be 21. Analysis of variance (ANOVA) with Dunn s correction was used to estimate and compare data for viral and bacterial titers, weight loss, cell counts, and cytokine levels. Paired Student s t tests were used to compare matched groups of mice for weight loss results in Fig. 7. A P value of 0.05 was considered significant for these comparisons. SigmaStat for Windows (SysStat Software, Inc.) (version 3.11) was utilized for all statistical analyses. RESULTS Mapping of the proposed H3N2 PB1-F2 proinflammatory sequence. The sequences of the HK68 and Wuh95 PB1-F2 proteins differ at 5 positions (aa residues 62, 73, 75, 79, and 82) in the C-terminal third (aa 61 to 87) that had previously been demonstrated to be sufficient to induce inflammatory responses (Fig. 1) (18, 19). To establish the individual roles of these amino acid residues in PB1-F2 pathogenicity, we synthesized HK68 or Wuh95 PB1-F2-derived peptides possessing single or multiple substitutions at these positions by replacing the sequence of interest with the corresponding sequence from the corresponding PB1-F2 ORF of the other virus (see Materials and Methods). The peptides were then administered a single time to groups of BALB/c mice (n 10) i.n. at a dosage of 60 mg per mouse. Mice exposed to the HK68 PB1-F2-derived peptide lost more than 20% of their initial weight (Fig. 2A), and 50% of them died within 21 days of peptide administration (Table 1). In contrast, exposure of mice to Wuh95 PB1-F2 caused no weight loss (Fig. 2B) or mortality (Table 1). Single amino acid substitutions at residues 62, 75, and 82 on HK68 and residues 75, 79, and 82 on Wuh95 PB1-F2 backgrounds significantly altered the properties of the natural peptides (reflected by

4 VOL. 85, 2011 ROLE OF H3N2 PB1-F2 IN INFLUENZA PATHOGENESIS FIG. 2. Weight loss of mice exposed to H3N2 PB1-F2-derived peptides. C-terminal peptides derived from the PB1-F2 sequences of HK68 (A) and Wuh95 (B) with the indicated alterations to the amino acid sequence were administered to groups of BALB/c mice (n 10), and mice were monitored individually for weight loss; results are presented as mean percentages of starting weight standard deviations (SD). HK68-4 contained the L62P, R75H, R79Q, and L82S changes, whereas Wuh95 4 had the P62L, H75R, Q79R, and S82L changes. An asterisk indicates a significant (P 0.05) difference in weight data on the indicated day for groups exposed to altered peptides compared to the results determined for the group exposed to the wild-type sequence of the HK68 (A) or Wuh95 (B) peptide. either weight loss or survival; P 0.05). Mutation at aa residue 73 in either background did not cause a statistically significant effect. The most prominent alterations in weight and mortality for mice were observed with Wuh95 and HK68 PB1-F2 peptides possessing quadruple mutations at aa 62, 75, 79, and 82 (the Wuh95 4 and HK68-4 peptides, respectively); the combination of these four changes was sufficient in either background to cause reversion of the phenotype (Fig. 2A and B; Table 1). Inflammatory responses in mice caused by H3N2 PB1-F2 peptides. To determine the roles of aa residues 62, 75, 79, and 82 in PB1-F2-mediated inflammation, we examined the lungs of mice for histopathologic changes at day 3 after peptide administration. Extensive lung consolidation with dense neutrophilic infiltrates in a peribronchiolar distribution and abundant monocytes and macrophages were observed in the parenchyma in lungs of mice exposed to 40 mg of Wuh95 4 or HK68 PB1-F2 peptides (Fig. 3A, E, and F; L62, R75, R79, and L82 amino acid sequences). There were no signs of lung inflammation in the control group that received PBS only (Fig. 3C), or in the groups of mice exposed to the Wuh95 and HK68-4 PB1-F2 peptides (Fig. 3B, D, and F; P62, H75, Q79, and S82 amino acid sequences), as determined by gross examination or by semiquantitative scoring of histopathology (Fig. 3F). Quantification of the immune cells and cytokines present in BAL fluid showed that the extensive pulmonary inflammation observed in mice exposed to Wuh95 4 and HK68 PB1-F2 peptides results from an influx of WBCs into the BAL fluid (Fig. 4A), as characterized by a significant increase in levels of neutrophils (Fig. 4B), macrophages (Fig. 4C), and cytokines (Fig. 4D to H), including IL-6, KC, MCP-1, MIP-1, and G-CSF, compared to those in PBS-, Wuh95-, or HK68-4- treated groups (P 0.05). Cytokine responses measured in the J774 macrophage cell line treated with H3N2 PB1-F2 peptides for 4 h at dosages ranging from 100 M to500 M revealed an increase of more than 20-fold in the levels of TNF- in culture supernatant fluids in cells treated with 250 M or500 M Wuh95 4 and HK68 compared to those treated with Wuh95 and HK68-4 PB1-F2 peptides (P 0.05; Fig. 5A and B). We conclude from these data that the presence of the HK68 amino acid motif at positions 62, 75, 79, and 82 is sufficient to allow PB1-F2-derived peptides to cause inflammation and that those inflammatory responses can contribute to morbidity and mortality in mice independently of other virulence factors expressed by the virus. The inflammatory motif of the H3N2 PB1-F2 peptide primes for bacterial pneumonia. We previously showed that PB1-F2 can accelerate development of secondary bacterial pneumonia in mice (13, 19). Whether this was dependent on the inflammatory activity of the protein was unclear. To establish the role of the H3N2 PB1-F2 proinflammatory motif in priming for bacterial pneumonia, we treated mice with 40 mg of a panel of TABLE 1. Survival of mice exposed to H3N2 PB1-F2-derived peptides a PB1-F2 peptide Mean no. of days to death SD Survival (%) No. of survivors/total no. of mice Wuh /10 P62L /10 R73K /10 H75R /10 Q79R /10 S82L /10 P62L, H75R, Q79R, S82L /10 HK /10 L62P /10 b K73R /10 R75H /10 b R79Q /10 L82S /10 b L62P, R75H, Q79R, L82S /10 b a BALB/c mice intranasally exposed to 60 mg of H3N2 PB1-F2 peptides per mouse were monitored for 21 days to determine the number of mice that died and the mean number of days to death. Control mice were treated only with PBS (data not shown). b The number of mice that survived exposure to peptide with mutations differed significantly from the number of those that survived exposure to naturally occurring peptide, as compared by the Kaplan-Meier method followed by a log-rank test.

5 12328 ALYMOVA ET AL. J. VIROL. PB1-F2 peptides and 1 day later challenged the mice with 2,000 CFU of S. pneumoniae. Weight loss and survival of mice were monitored for 21 days after the bacterial challenge. Mice exposed to the proinflammatory HK68 and Wuh95 4 PB1-F2 peptides lost about 15% of their initial weight after peptide administration (Fig. 6A and B). Subsequent challenge with S. pneumoniae enhanced this weight loss and had a significant (P 0.05) effect on mortality (Fig. 6A to D). All mice exposed to the HK68 peptide and challenged with PBS (instead of bacteria) survived, while 40% died by day 3 after subsequent infection with S. pneumoniae (Fig. 6C). We observed 20% mortality in the group of mice exposed to Wuh95 4 peptide and challenged with PBS (Fig. 6D), and all mice in the Wuh95 4 group that were challenged with bacteria died by day 5 after peptide administration. The most striking differences in weight loss and survival were seen in comparisons of the groups of mice exposed to the H3N2 PB1-F2 peptides with (Wuh95 4 and HK68) and without (Wuh95 and HK68-4) proinflammatory motifs in the bacterial pneumonia model. Mice in the latter two groups lost an average of only 5% of their initial weight after peptide administration, did not lose further weight upon bacterial challenge, and did not succumb to infection. Interestingly, the weight loss of mice exposed to PBS (instead of peptide) and bacteria was higher by day 7 after peptide administration than that seen with the groups of mice exposed to noninflammatory peptides (Wuh95 and HK68-4) and bacteria, and the proinflammatory effects of the Wuh95 4 peptide appeared to be greater than the effects of the HK68 peptide, despite HK68 being the original wild-type pandemic strain. Examination of the mouse lung bacterial titers at days 1, 3, and 5 after pneumococcal challenge indicated that the excessive weight loss and mortality observed in groups of mice exposed to the proinflammatory peptides (Wuh95 4 and HK68) were due to an increase of up to 70-fold in bacterial numbers compared to the results seen with groups of mice that were subjected to mock treatment with PBS (P 0.05; Fig. 6E and F). Surprisingly, the bacterial lung titers in PBS-treated group were significantly higher than those in groups of mice exposed to Wuh95 and HK68-4, suggesting that the noninflammatory variants of the H3N2 PB1-F2 peptides have antibacterial properties (P 0.05). This proposed antibacterial effect correlated with the modest differences in weight loss evident in comparisons of the groups administered PBS and bacteria to the groups administered noninflammatory peptide and bacteria (Fig. 6A and B). To confirm that the effect of the observed inhibitory activity of the Wuh95 PB1-F2 peptide on S. pneumoniae was a direct effect, approximately CFU/ml of bacteria were treated with increasing doses of Wuh95 and Wuh95 4 peptides. Bacterial colony counts showed a reduction of S. pneumoniae growth resulting from treatment with Wuh95 compared to the FIG. 3. Histopathologic changes in the lungs of mice exposed to H3N2 PB1-F2-derived peptides. BALB/c mice were exposed to peptides derived from HK68 (A) or Wuh95 (B), a version of HK68 altered at L62P, R75H, R79Q, and L82S (HK68-4) (D), a version of Wuh95 altered at P62L, H75R, Q79R, and S82L (Wuh95 4) (E), or PBS (C). Sections of lungs stained with hematoxylin and eosin are pictured at 20 magnification. (F) A semiquantitative grading system was used to assess the degree of lung injury present in each set of lungs. The scores (means SD) determined for 3 mice per group are shown. An asterisk indicates a significant (P 0.05) difference by ANOVA compared to the results determined for the PBS, Wuh95, and HK68-4 groups.

6 VOL. 85, 2011 ROLE OF H3N2 PB1-F2 IN INFLUENZA PATHOGENESIS FIG. 4. Inflammatory responses in mice exposed to H3N2 PB1-F2- derived peptides. The numbers (means SD) of total white blood cells (WBCs) (A), neutrophils (B), and macrophages (C) recovered from bronchoalveolar lavage (BAL) fluid are compared for groups of BALB/c mice (n 4 or 5) exposed to PBS (as a control) or PB1-F2 peptides derived from HK68, Wuh95, a version of HK68 altered at L62P, R75H, R79Q, and L82S (HK68-4), or a version of Wuh95 altered at P62L, H75R, Q79R, and S82L (Wuh95 4). (D to H) The concentrations (means SD) of cytokines and chemokines, including G-CSF, IL-6, KC, MCP-1, and MIP-1, were determined from the same BAL fluids. An asterisk indicates a significant (P 0.05) difference by ANOVA compared to the results determined for the PBS, Wuh95, and HK68-4 groups. results seen with untreated bacteria (see Fig. 8A). The addition of the Wuh95 4 peptide to S. pneumoniae bacterial cultures did not cause bactericidal effects at doses below 500 M. These results support our in vivo data suggesting that the Wuh95 PB1-F2 possesses direct antibacterial activity against S. pneumoniae. The results from these experiments performed with H3N2 PB1-F2-derived peptides indicate that the presence of aa residues L62, R75, R79, and L82 causes significant pulmonary inflammation and accelerates development of pneumococcal infection. The presence of aa residues P62, H75, Q79, and S82 on H3N2 PB1-F2 does not induce inflammation but has an apparent bactericidal effect on S. pneumoniae. Role of the H3N2 PB1-F2 inflammatory motif in virus pathogenicity. To determine the role of aa residues 62, 75, 79, and 82 in virus pathogenicity in mice, we attempted to rescue chimeric viruses carrying six genes from Wuh95 and the HA and NA genes from PR8 without or with mutations in Wuh95 PB1-F2. However, in the case of aa residue 79, the primary FIG. 5. Inflammatory response of J774 macrophages to H3N2 PB1- F2-derived peptides. J774 macrophages were exposed to PBS (as a control) or peptides derived from HK68 (A) or Wuh95 (B) at a concentration of 100, 250, or 500 M. Four hours later, the concentration of TNF- was determined from the supernatants; the data are expressed as the means SD of the results of 3 independent experiments. HK68-4 is a version of HK68 altered at L62P, R75H, R79Q, and L82S, and Wuh95 4 is a version of Wuh95 altered at P62L, H75R, Q79R, and S82L. An asterisk indicates a significant (P 0.05) difference by ANOVA compared to the results determined for the other groups at that peptide concentration. PB1 amino acid sequence would have been altered, and that virus could not be rescued. Thus, we performed the studies with the chimeric Wuh95/PR8 strain (aa sequence P62, H75, and S82 with respect to the PB1-F2 ORF) and a virus possessing triple mutations of P62L, H75R, and S82L in Wuh95 PB1-F2 (Wuh95 3/PR8). DBA/2 mice were infected with doses ranging from 10 4 to 10 6 PFU per mouse and monitored for weight loss (Fig. 7A) and mortality (Table 2). Our data indicate that, as a trend, mice infected with the chimeric Wuh95 3/PR8 virus possessing the proinflammatory P62L, H75R, and S82L mutations in PB1-F2 experienced greater weight loss and mortality than those infected with Wuh95/PR8. The clearest differences in the effects of the two viruses were observed at a dose of PFU per mouse. The mice infected with Wuh95 3/PR8 lost an average of 16.3% of their initial weight by day 7 p.i. and 40% of them died by day 10 p.i. versus the 10.2% of weight loss by day 7 p.i. and 20% mortality observed for Wuh95/PR8 (Fig. 7A; Table 2). Differences in weight loss reached statistical significance (P 0.05) by day 7 p.i. for the groups infected with and PFU per mouse. Examination of the growth kinetics of Wuh95/PR8 and Wuh95 3/PR8 in the lungs of mice infected with PFU per mouse showed that, despite the fact that the mice exhibited greater rates of weight loss and mortality, the growth of Wuh95 3/PR8 in mouse lungs was similar to or modestly lower than that of Wuh95/PR8 up to day 3 p.i. (Fig. 7B). We observed 5-to-10-fold differences in lung virus titers between the two groups, with the greatest differences observed at 12 h after infection. An examination of the levels of WBCs, macrophages, neutrophils, and inflammatory dendritic cells as well as histopathology results and the levels of proinflammatory cytokines determined using the BAL fluid of mice infected with

7 12330 ALYMOVA ET AL. J. VIROL. Downloaded from FIG. 6. Effect of H3N2 PB1-F2-derived peptides on bacterial pneumonia. Weight loss (A and B), survival (C and D), and bacterial lung titers (E and F) of groups of mice (n 8 to 10) exposed to PB1-F2 peptides derived from HK68 (A, C, and E) or Wuh95 (B, D, and F) are shown in a bacterial pneumonia model. HK68-4 is a version of HK68 altered at L62P, R75H, R79Q, and L82S, and Wuh95 4 is a version of Wuh95 altered at P62L, H75R, Q79R, and S82L. BALB/c mice were exposed to H3N2 PB1-F2 peptides or PBS and then challenged 1 day later with a dose of 2,000 CFU of S. pneumoniae (SPn) per mouse. (E) Lungs from 3 to 5 mice per group were collected at days 1, 3, and 5 after bacterial challenge and titrated for bacterial load. Error bars indicate the SD of the means. An asterisk indicates a significant (P 0.05) difference compared to the results determined for the PBS SPn, HK68-4 PBS, and HK68-4 SPn groups (A and B), all other groups (C and D), the HK68-4 group (E), and the Wuh95 group (F) by ANOVA (A, B, E, and F) and the Kaplan-Meier method followed by a log-rank test (C and D). A double asterisk indicates a significant difference in weight loss (A) and in bacterial titers (F) compared to the results determined for all other groups. Wuh95/PR8 and Wuh95 3/PR8 at PFU per mouse at days 3, 7, and 9 p.i. did not reveal significant differences in these parameters between the groups at those time points (data not shown). Thus, neither improved replication nor pathological changes in the lungs could be positively linked to the observed pathogenic phenotype of the mutant virus in this model. The proinflammatory motif of the 1968 H3N2 PB1-F2 protein primes for secondary bacterial pneumonia in the context of the full virus. To determine the role of the Wuh95 3/PR8 PB1-F2 proinflammatory residues in promoting secondary bacterial pneumonia, we developed a nonlethal synergistic model using chimeric viruses and S. pneumoniae by infecting groups of 10 mice with a single dose of PFU of Wuh95/PR8 or Wuh95 3/PR8 per mouse and, 7 days later, challenging the groups with 100 CFU of bacteria per mouse. The differences between the two chimeric viruses were evaluated by measuring weight loss and bacterial and virus lung titers. Infecting mice with either virus at the chosen dose did not result in significant weight loss during the observation period (mice weighed 99.0% and 98.8% of their starting weights at day 7 for the Wuh95/PR8 and Wuh95 3/PR8 groups, respectively).viral lung titers were similar for the Wuh95/PR8 and Wuh95 3/PR8 groups ( PFU/ml and PFU/ ml, respectively) at the time of secondary bacterial challenge (day 7 after viral infection). Bacterial superinfection notably increased the weight loss only in the group of mice infected with Wuh95 3/PR8 (Fig. 8B; 96.0% of starting weight versus on November 7, 2018 by guest

8 VOL. 85, 2011 ROLE OF H3N2 PB1-F2 IN INFLUENZA PATHOGENESIS FIG. 7. Characterization of PB1-F2 chimeric viruses in the Wuh95 background. (A) Groups of DBA/2 mice (n 10) were infected with wild-type Wuh95/PR8 or an isogenic mutant of PB1-F2 altered at P62L, H75R, and S82L (Wuh95 3/PR8) at a dose of 10 4 (4), (4.5), 10 5 (5), or 10 6 (6) PFU per mouse and monitored for weight loss. (B) Growth kinetics of those viruses for a dose of PFU per mouse were determined in groups of mice (n 3 to 5 per time point). Error bars indicate the SD. An asterisk indicates a significant (P 0.05) difference for the mutant virus compared to the results determined for the wild-type strain. FIG. 8. Interactions of Wuh95 PB1-F2 with bacteria. (A) Bacterial cultures (n 4 per dose and time point) were incubated for 6 h with PBS or wild-type Wuh95 peptide, Wuh95 4 peptide, or melittin as a positive control at doses escalating from 1 to 500 M, and titers were determined by plating serial dilutions. (B) Groups of mice (n 10) were infected with wild-type Wuh95/PR8 (Wuh95) or an isogenic mutant of PB1-F2 altered at P62L, H75R, and S82L (Wuh95 3) and then challenged 7 days later with either PBS or 100 CFU of S. pneumoniae and monitored for weight loss. Error bars indicate the SD. 90.5% for the Wuh95/PR8 and Wuh95 3/PR8 groups, respectively) (P 0.05). No virus was present in the lungs of mice in either group at day 2 after the bacterial challenge (day 9 after viral infection). Analysis of bacterial lung titers at this time point showed no detectable titers in the groups of mice infected with Wuh95/PR8 or exposed to PBS (instead of virus). In contrast, the mean level of bacterial lung titers for the group of mice infected with Wuh95 3/PR8 was CFU/ml (P 0.05). This result was similar to the data obtained with Wuh95 and Wuh95 4 PB1-F2 peptides in the bacterial pneumonia model. We conclude from these data that, in the context of the full virus, the pathogenic effects of H3N2 PB1-F2 protein are manifested primarily through interactions with bacteria. The proinflammatory properties of 1968 pandemic strain PB1-F2 support bacterial superinfection, whereas the antibacterial properties of Wuh95 PB1-F2 suppress bacterial growth in the lung. DISCUSSION Since their emergence in 1968, H3N2 subtype IAVs have been the primary cause of excess mortality among influenza viruses (8, 23). However, both influenza like-illness and excess mortality have declined since the mid-1980s compared to the first 2 decades of circulation of this new subtype (8, 9, 23). The specific mechanisms underlying this diminution of disease are Virus dose (PFU/mouse) No. of survivors/total no. of mice TABLE 2. Survival of mice infected with chimeric viruses a Wuh95/PR8 Survival (%) Mean no. of days to death SD No. of survivors/total no. of mice Wuh95 3/PR8 Survival (%) Mean no. of days to death SD / / / / / / / / a DBA/2 mice infected with different doses of chimeric viruses were monitored for 21 days to determine the number of mice that died and the mean number of days to death. Control mice were treated only with PBS (data not shown).

9 12332 ALYMOVA ET AL. J. VIROL. unclear, but it is likely that changes to multiple virulence factors of the virus during adaptation of genes to the human host are involved. Only two gene segments, the HA and PB1, were new in the pandemic strain of 1968; those are therefore prime candidates for these hypothesized adaptive changes. The sequential addition of sites for N-linked glycosylation to the globular head of the HA, shifting tropism to the upper respiratory tract by allowing clearance by collagenous lectins, has been proposed as one factor explaining the decreased virulence of recent seasonal H3N2 viruses (25). Here we propose that a change to the PB1-F2 protein, which is encoded by the PB1 gene segment, is another factor. Taking advantage of prior work demonstrating that the proinflammatory potential of the PB1-F2 proteins from the 1968 pandemic strain differed from that of the PB1-F2 proteins of Wuh95, a recent seasonal strain (18), we have mapped the relevant sequence motif in the H3N2 lineage. Amino acid residues L62, R75, R79, and L82 in the H3N2 background are sufficient for the generation of inflammatory responses to the C terminus of PB1-F2, including elevated proinflammatory cytokine levels and an influx of neutrophils and macrophages into the lungs and airways, with resulting pathological damage, weight loss, and death. The motif consisting of L62, R75, R79, and L82 is also present in the PB1-F2 proteins of other epidemiologically important strains, including the 1918 (H1N1) and 1957 (H2N2) pandemic strains, and in many highly pathogenic avian influenza viruses of the H5N1 subtype (18). However, there are several other sequence differences in PB1-F2 in those influenza viruses, so whether those specific amino acids are either necessary or sufficient for the demonstrated proinflammatory activity of those PB1-F2 proteins (18) remains an open question. The transition in amino acid sequence from the full proinflammatory motif to the Wuh95 sequence began in 1972 with an L82S mutation, followed in 1974 by R79Q, in 1987 by R75H, and in 1995 by L62P (Fig. 1). This parallels the general decline in severe outcomes associated with these strains seen over several decades (8, 9). The lack of a strong proinflammatory phenotype attributable to PB1-F2 in the context of the full influenza virus suggests that alterations to primary viral virulence are not responsible for any theoretical contributions of PB1-F2 to that epidemiologic pattern. This fits with prior data from our laboratory suggesting that deletion of PB1-F2 from seasonal strains such as PR8 does not have a strong phenotype (19, 20) but that PB1-F2 may play a more important role in the primary pathogenesis of specific highly pathogenic influenza viruses such as the 1918 and H5N1 strains (6, 7, 18, 19, 28). However, the experiments performed with deletion mutants may be confounded by altered regulation of PB1 and N40, making firm conclusions difficult (26). The approach taken here on the basis of mutation of the full-length protein may better explicate the true contribution of the protein. However, as we were unable to rescue an otherwise isogenic virus with all 4 mutations, the possibility remains that versions of PB1-F2 could contribute to viral virulence, as has been documented for the 1918 and H5N1 strains. Since most excess mortality from seasonal influenza viruses is due to secondary bacterial infections or interactions with comorbidities such as heart and lung disease (4, 21), the ability of the proinflammatory PB1-F2 proteins to prime for bacterial pneumonia may be a more relevant mechanism than proposed changes to viral virulence to explain the contribution of the PB1-F2 protein to the pathogenesis of seasonal variants. The idea suggested by our data that certain PB1-F2 proteins may actually have antibacterial properties is interesting but was not entirely unexpected. Structurally, the C-terminal portion of PB1-F2 is proposed to form an alpha helix in a membraneassociated environment (1). This region is also highly cationically charged, and, like other small peptides with antimicrobial activity, can induce pore formation in membranes (2), leading to the reasonable supposition that it could disrupt and kill bacteria. Thus, the finding that the PR8 and 1918 PB1-F2 proteins supported secondary bacterial infections rather than preventing them was initially a surprise to us (19). In the context of evolution and adaptation to the mammalian lung, however, it is possible that the proinflammatory properties of recent PB1-F2 émigrés from the avian reservoir are detrimental to the virus and that a transition over time from an inflammatory function to an antibacterial function would be of benefit. We hypothesize that this ability represents a form of viral-bacterial warfare directed against respiratory bacteria that might interfere with the host niche of IAVs. However, these data need to be confirmed and extended before this can be accepted as a relevant function in viral adaptation or mitigation of disease. In the context of a future pandemic, our data suggest that surveillance and prioritization of strains should particularly focus on those that express a full-length PB1-F2 protein with the proinflammatory sequence motif described in this work. The 2009 pandemic H1N1 strain took its PB1 gene segment from a human H3N2 virus some time in the late 1990s or early 2000s (22) and has a mixed pattern of predicted residues of L62, H75, Q79, and L82 at those positions but stop codons at positions 12 and 57 such that the C-terminal portion of the protein is not expressed. Hai et al. have shown that reversion of these stop codons to allow expression of the full-length protein has minimal effects on primary viral pathogenesis in animals as measured by weight loss, mortality, or viral titers (11). These data suggested that further mutations would be necessary to achieve enhanced viral virulence through PB1-F2- related mechanisms. However, many strains from diverse IAV subtypes currently circulating in both the avian and swine reservoirs express full-length PB1-F2 proteins that contain the full proinflammatory motif (data not shown). Those strains may represent a threat to cause a more severe pandemic either through the enhancement of primary viral virulence as is seen with 1918 and H5N1 strains (7, 19) or through the potentiation of secondary bacterial disease. ACKNOWLEDGMENTS This work was supported by research grant AI from the National Institutes of Health and by the American Lebanese Syrian Associated Charities (ALSAC). We acknowledge helpful discussions with Jonathan Yewdell (Laboratory of Viral Diseases, National Institutes of Health) and technical assistance from Amy Iverson. REFERENCES 1. Bruns, K., et al Structural characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein. J. Biol. Chem. 282: Chanturiya, A. N., et al PB1-F2, an influenza A virus-encoded pro-

10 VOL. 85, 2011 ROLE OF H3N2 PB1-F2 IN INFLUENZA PATHOGENESIS apoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes. J. Virol. 78: Chen, W., et al A novel influenza A virus mitochondrial protein that induces cell death. Nat. Med. 7: Collins, S. D Excess mortality from causes other than influenza and pneumonia during influenza epidemics. Public Health Rep. 47: Condit, R. C Virus cultivation, detection, and genetics, p In S. J. Flint, L. W. Enquist, R. M. Krug, V. R. Racaniello, and T. Skarzynski (ed.), Principles of virology: molecular biology, pathogenesis, and control of animal viruses. ASM Press, Washington, DC. 6. Conenello, G. M., et al A single N66S mutation in the PB1-F2 protein of influenza A virus increases virulence by inhibiting the early interferon response in vivo. J. Virol. 85: Conenello, G. M., D. Zamarin, L. A. Perrone, T. Tumpey, and P. Palese A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence. PLoS Pathog. 3: Doshi, P Trends in recorded influenza mortality: United States, Am. J. Public Health 98: Fleming, D. M., and A. J. Elliot Lessons from 40 years surveillance of influenza in England and Wales. Epidemiol. Infect. 136: Glaser, L., et al A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity. J. Virol. 79: Hai, R., et al PB1-F2 expression by the 2009 pandemic H1N1 influenza virus has minimal impact on virulence in animal models. J. Virol. 84: Hoffmann, E., S. Krauss, D. Perez, R. Webby, and R. G. Webster Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine 20: Iverson, A. R., et al Influenza virus primes mice for pneumonia from Staphylococcus aureus. J. Infect. Dis. 203: Kash, J. C., et al Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature 443: Kawaoka, Y., S. Krauss, and R. G. Webster Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J. Virol. 63: Kilbourne, E. D Influenza pandemics in perspective. JAMA 237: Mazur, I., et al The proapoptotic influenza A virus protein PB1-F2 regulates viral polymerase activity by interaction with the PB1 protein. Cell. Microbiol. 10: McAuley, J. L., et al PB1-F2 proteins from H5N1 and 20 century pandemic influenza viruses cause immunopathology. PLoS Pathog. 6:e McAuley, J. L., et al Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia. Cell Host Microbe 2: McAuley, J. L., K. Zhang, and J. A. McCullers The effects of influenza A virus PB1-F2 protein on polymerase activity are strain specific and do not impact pathogenesis. J. Virol. 84: McCullers, J. A Insights into the interaction between influenza virus and pneumococcus. Clin. Microbiol. Rev. 19: Peiris, J. S., L. L. Poon, and Y. Guan Emergence of a novel swineorigin influenza A virus (S-OIV) H1N1 virus in humans. J. Clin. Virol. 45: Reichert, T. A., et al Influenza and the winter increase in mortality in the United States, Am. J. Epidemiol. 160: Smith, M. W., J. E. Schmidt, J. E. Rehg, C. Orihuela, and J. A. McCullers Induction of pro- and anti-inflammatory molecules in a mouse model of pneumococcal pneumonia following influenza. Comp. Med. 57: Vigerust, D. J., et al N-linked glycosylation attenuates H3N2 influenza viruses. J. Virol. 81: Wise, H. M., et al A complicated message: identification of a novel PB1-related protein translated from influenza A segment 2 mrna. J. Virol. 83: Zamarin, D., A. Garcia-Sastre, X. Xiao, R. Wang, and P. Palese Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1. PLoS Pathog. 1:e Zamarin, D., M. B. Ortigoza, and P. Palese Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice. J. Virol. 80: Downloaded from on November 7, 2018 by guest

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