The influenza virus protein PB1-F2 inhibits the induction of type I interferon at the level of the MAVS adaptor protein

PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses and has been shown to contribute to viral pathogenicity. Notably, a serine at position 66 (66S) in PB1-F2 is known to increase virulence compared to an isogenic virus with an asparagine (66N) at this position. Recently, we found that an influenza virus expressing PB1-F2 N66S suppresses interferon (IFN)-stimulated genes in mice. To characterize this phenomenon, we employed several in vitro assays. Overexpression of the A/Puerto Rico/8/1934 (PR8) PB1-F2 protein in 293T cells decreased RIG-I mediated activation of an IFN-β reporter and secretion of IFN as determined by bioassay. Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype. Similar observations were found in the context of viral infection with a PR8 PB1-F2 N66S virus. To understand the relationship between NS1, a previously described influenza virus protein involved in suppression of IFN synthesis, and PB1-F2, we investigated the induction of IFN when NS1 and PB1-F2 were co-expressed in an in vitro transfection system. In this assay we found that PB1-F2 N66S further reduced IFN induction in the presence of NS1. By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS). Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS. In summary, we have characterized the anti-interferon function of PB1-F2 and we suggest that this activity contributes to the enhanced pathogenicity seen with PB1-F2 N66S- expressing influenza viruses.     

Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20(th) century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism.     

A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence

The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N-infected mice compared with wild-type 1918 virus-infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus.     

In silico thermodynamic stability of mammalian adaptation and virulence determinants in polymerase complex proteins of H9N2 virus

The polymerase complex proteins (PB2, PB1, and PA) are responsible primarily for the replication of avian influenza virus and play an important role in virus virulence, mammalian adaptation, and interspecies transmission. In this study; eight Egyptian LPAI-H9N2 viruses isolated from apparent healthy chickens and quails from 2014 to 2016. Characterization of complete nucleotide sequences, phylogenetic and mutation analysis were carried out. The measurement of thermodynamic stability of the H9N2 polymerase protein in comparison to human H3N2 and H1N1 proteins was carried out using in silico method. Phylogenetic analysis of these viruses revealed a close relationship to viruses isolated from neighboring Middle Eastern countries with an average of 96–99% homology. They are sharing the common ancestor A/quail/Hong Kong/G1/1997 (G1-Like) without any evidence for genetic reassortment. In addition, eight markers related to virulence were identified, including the combination of 627V and 391E in the PB2 gene with full-length PB1-F2 and PA-X proteins were observed in all viruses and the substitution N66S in PB1-F2 which suggest increasing virus virulence. Moreover, six markers that may affect the virus replication and transmission in mammalian hosts were identified. Five mutations related to mammalian adaptation show a structural stabilizing effect on LPAI-H9N2 polymerase complex protein according to the free-energy change (ΔΔG). Three out of those six adaptive mutations shown to increase polymerase complex protein stability were found in Egyptian LPAI-H9N2 viruses similar to Human H3N2 and H1N1 (661 in PB2, 225 and 409 in PA genes). Our results suggested that the stabilizing mutations in the polymerase complex protein have likely affected the protein structure and induced favorable conditions for avian virus replication and transmission in mammalian hosts. Indeed, the study reports the mutational analysis of the circulating LPAI-H9N2 strains in Egypt.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.     

Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens

A combination of viral, bacterial, and host factors contributes to the severity and overall mortality associated with influenza virus-bacterium superinfections. To date, the virulence associated with the recently identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae superinfection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a nonhuman host species (swine). Using this rationale, we developed the hypothesis that naturally occurring viruses expressing variants of genes, like the PB1-F2 gene, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from superinfection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial superinfection, are broadly associated with enhanced pathogenicity in swine in a bacterium-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with Streptococcus pyogenes superinfection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of superinfections with specific Gram-positive respiratory pathogens.     

PB1-F2 modulates early host responses but does not affect the pathogenesis of H1N1 seasonal influenza virus

In the context of infections with highly pathogenic influenza A viruses, the PB1-F2 protein contributes to virulence and enhances lung inflammation. In contrast, its role in the pathogenesis of seasonal influenza viral strains is less clear, especially in the H1N1 subtype, where strains can have a full-length 87- to 90-amino-acid protein, a truncated 57-amino-acid version, or lack the protein altogether. Toward this, we introduced the full-length 1918 PB1-F2, or prevented PB1-F2 expression, in H1N1 A/USSR/90/77, a seasonal strain that naturally expresses a truncated PB1-F2. All viruses replicated with similar efficiency in ferret or macaque ex vivo lung cultures and elicited similar cytokine mRNA profiles. In contrast, the virus expressing the 1918 PB1-F2 protein caused a delay of proinflammatory responses in ferret blood-derived macrophages, while the PB1-F2 knockout virus resulted in a more rapid response. A similar but less pronounced delay in innate immune activation was also observed in the nasal wash cells of ferrets infected with the 1918 PB1-F2-expressing virus. However, the three viruses did not differ in their virulence or clinical course in ferrets, supporting speculations that PB1-F2 is of limited importance for the pathogenesis of primary viral infection with human seasonal H1N1 viruses.     

PB1-F2 Proteins from H5N1 and 20th Century Pandemic Influenza Viruses Cause Immunopathology

With the recent emergence of a novel pandemic strain, there is presently intense interest in understanding the molecular signatures of virulence of influenza viruses. PB1-F2 proteins from epidemiologically important influenza A virus strains were studied to determine their function and contribution to virulence. Using 27-mer peptides derived from the C-terminal sequence of PB1-F2 and chimeric viruses engineered on a common background, we demonstrated that induction of cell death through PB1-F2 is dependent upon BAK/BAX mediated cytochrome c release from mitochondria. This function was specific for the PB1-F2 protein of A/Puerto Rico/8/34 and was not seen using PB1-F2 peptides derived from past pandemic strains. However, PB1-F2 proteins from the three pandemic strains of the 20^th century and a highly pathogenic strain of the H5N1 subtype were shown to enhance the lung inflammatory response resulting in increased pathology. Recently circulating seasonal influenza A strains were not capable of this pro-inflammatory function, having lost the PB1-F2 protein''s immunostimulatory activity through truncation or mutation during adaptation in humans. These data suggest that the PB1-F2 protein contributes to the virulence of pandemic strains when the PB1 gene segment is recently derived from the avian reservoir.     

PB1-F2 amyloid-like fibers correlate with proinflammatory signaling and respiratory distress in influenza-infected mice

PB1-F2 is a virulence factor of influenza A virus known to increase viral pathogenicity in mammalian hosts. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers. However, the correlation between PB1-F2 structures and the resulting inflammatory response is unknown. Here, we used synchrotron-coupled Fourier transform-IR and deep UV microscopies to determine the presence of PB1-F2 fibers in influenza A virus–infected mice. In order to study the correlation between PB1-F2 structure and the inflammatory response, transgenic mice expressing luciferase under the control of an NF-κB promotor, allowing in vivo monitoring of inflammation, were intranasally instilled with monomeric, fibrillated, or truncated forms of recombinant PB1-F2. Our intravital NF-κB imaging, supported by cytokine quantification, clearly shows the proinflammatory effect of PB1-F2 fibers compared with N-terminal region of PB1-F2 unable to fibrillate. It is noteworthy that instillation of monomeric PB1-F2 of H5N1 virus induced a stronger inflammatory response when compared with prefibrillated PB1-F2 of H1N1 virus, suggesting mechanisms of virulence depending on PB1-F2 sequence. Finally, using whole-body plethysmography to measure volume changes in the lungs, we quantified the effects of the different forms of PB1-F2 on respiratory parameters. Thus, we conclude that PB1-F2–induced inflammation and respiratory distress are tightly correlated with sequence polymorphism and oligomerization status of the protein.     

Influenza A Virus PB1-F2 Protein Expression Is Regulated in a Strain-Specific Manner by Sequences Located Downstream of the PB1-F2 Initiation Codon

Translation of influenza A virus PB1-F2 occurs in a second open reading frame (ORF) of the PB1 gene segment. PB1-F2 has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis. Experimental evidence of PB1-F2 molecular function during infection has been collected primarily from human and avian viral isolates. As the 2009 H1N1 (H1N1pdm09) strain highlighted, some swine-derived influenza viruses have the capacity to infect human hosts and emerge as a pandemic. Understanding the impact that virulence factors from swine isolates have on both human and swine health could aid in early identification of viruses with pandemic potential. Studies examining PB1-F2 from swine isolates have focused primarily on H1N1pdm09, which does not encode PB1-F2 but was engineered to carry a full-length PB1-F2 ORF to assess the impact on viral replication and pathogenicity. However, experimental evidence of PB1-F2 protein expression from swine lineage viruses has not been demonstrated. Here, we reveal that during infection, PB1-F2 expression levels are substantially different in swine and human influenza viruses. We provide evidence that PB1-F2 expression is regulated at the translational level, with very low levels of PB1-F2 expression from swine lineage viruses relative to a human isolate PB1-F2. Translational regulation of PB1-F2 expression was partially mapped to two independent regions within the PB1 mRNA, located downstream of the PB1-F2 start site. Our data suggest that carrying a full-length PB1-F2 ORF may not be predictive of PB1-F2 expression in infected cells for all influenza A viruses.     

Restored PB1-F2 in the 2009 pandemic H1N1 influenza virus has minimal effects in swine

PB1-F2 is an 87- to 90-amino-acid-long protein expressed by certain influenza A viruses. Previous studies have shown that PB1-F2 contributes to virulence in the mouse model; however, its role in natural hosts-pigs, humans, or birds-remains largely unknown. Outbreaks of domestic pigs infected with the 2009 pandemic H1N1 influenza virus (pH1N1) have been detected worldwide. Unlike previous pandemic strains, pH1N1 viruses do not encode a functional PB1-F2 due to the presence of three stop codons resulting in premature truncation after codon 11. However, pH1N1s have the potential to acquire the full-length form of PB1-F2 through mutation or reassortment. In this study, we assessed whether restoring the full-length PB1-F2 open reading frame (ORF) in the pH1N1 background would have an effect on virus replication and virulence in pigs. Restoring the PB1-F2 ORF resulted in upregulation of viral polymerase activity at early time points in vitro and enhanced virus yields in porcine respiratory explants and in the lungs of infected pigs. There was an increase in the severity of pneumonia in pigs infected with isogenic virus expressing PB1-F2 compared to the wild-type (WT) pH1N1. The extent of microscopic pneumonia correlated with increased pulmonary levels of alpha interferon and interleukin-1β in pigs infected with pH1N1 encoding a functional PB1-F2 but only early in the infection. Together, our results indicate that PB1-F2 in the context of pH1N1 moderately modulates viral replication, lung histopathology, and local cytokine response in pigs.     

1968−2014年中国甲型H3N2流感病毒PB1基因进化分析

【目的】分析季节性H3N2流感病毒PB1基因序列的变异情况,揭示H3N2流感病毒PB1基因的分子特征与进化趋势。【方法】对1968?2014年中国地区82株人H3N2毒株、2012?2014年江苏省分离的81株甲型H3N2流感病毒、6株SIV和4株AIV H3N2亚型PB1、PB1-F2基因进行分子进化分析。【结果】1968?2014年中国H3N2流感毒株PB1核苷酸和氨基酸相似性分别为90.91%?100%和96.91%?100%。系统进化树分析,1968?2014年共173株H3N2流感病毒总体上分为4个分支,2002?2014年分离毒株位于第IV分支上,1968?1994年分离毒株位于第II和III分支;猪源H3N2亚型分布于第I、II、IV分支上;分子特征显示PB1氨基酸52、113、179、216、576、586、619、621、709位在2002年以后发生适应性改变,替换了原来的氨基酸;PB1-F2基因编码截断型蛋白长度有52、34、25、24、11 aa (猪源),PB1-F2蛋白毒力关键位点上未出现高致病性特征突变。【结论】自1968年起H3N2亚型PB1基因变异逐步趋于稳定,且PB1-F2截断型毒株正逐渐成为一类新的进化特征,但PB1基因与其他亚型之间发生重配以及关键毒力位点的变异仍应是监测的重点。     

Influenza A virus protein PB1-F2 from different strains shows distinct structural signatures

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian influenza isolates. The structures of full-length PB1-F2 of the influenza strains Pandemic flu 2009 H1N1, 1918 Spanish flu H1N1, Bird flu H5N1 and H1N1 PR8, have been characterized by NMR and CD spectroscopy. The study was conducted using chemically synthesized full-length PB1-F2 protein and fragments thereof. The amino acid residues 30–70 of PR8 PB1-F2 were found to be responsible for amyloid formation of the protein, which could be assigned to formation of β-sheet structures, although α-helices were the only structural features detected under conditions that mimic a membranous environment. At membranous conditions, in which the proteins are found in their most structured state, significant differences become apparent between the PB1-F2 variants investigated. In contrast to Pandemic flu 2009 H1N1 and PR8 PB1-F2, which exhibit a continuous extensive C-terminal α-helix, both Spanish flu H1N1 and Bird flu H5N1 PB1-F2 contain a loop region with residues 66–71 that divides the C-terminus into two shorter helices. The observed structural differences are located to the C-terminal ends of the proteins to which most of the known functions of these proteins have been assigned. A C-terminal helix–loop–helix motif might be a structural signature for PB1-F2 of the highly pathogenic influenza viruses as observed for 1918 Spanish flu H1N1 and Bird flu H5N1 PB1-F2. This signature could indicate the pathological nature of viruses emerging in the future and thus aid in the recognition of these viruses.     

Immunopathogenic and antibacterial effects of H3N2 influenza A virus PB1-F2 map to amino acid residues 62, 75, 79, and 82

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.     

The Effects of Influenza A Virus PB1-F2 Protein on Polymerase Activity Are Strain Specific and Do Not Impact Pathogenesis

The influenza A virus PB1-F2 protein has been implicated as a virulence factor, but the mechanism by which it enhances pathogenicity is not understood. The PB1 gene segment of the H1N1 swine-origin influenza virus pandemic strain codes for a truncated PB1-F2 protein which terminates after 11 amino acids but could acquire the full-length form by mutation or reassortment. It is therefore important to understand the function and impact of this protein. We systematically assessed the effect that PB1-F2 expression has on viral polymerase activity, accumulation and localization of PB1, and replication in vitro and in mice. We used both the laboratory strain PR8 and a set of viruses engineered to study clinically relevant PB1-F2 proteins. PB1-F2 expression had modest effects on polymerase activity, PB1 accumulation, and replication that were cell type and virus strain dependent. Disruption of the PB1-F2 reading frame in a recent, seasonal H3N2 influenza virus strain did not affect these parameters, suggesting that this is not a universal function of the protein. Disruption of PB1-F2 expression in several backgrounds or expression of PB1-F2 from the 1918 pandemic strain or a 1956 H1N1 strain had no effect on viral lung loads in mice. Alternate mechanisms besides alterations to replication are likely responsible for the enhanced virulence in mammalian hosts attributed to PB1-F2 in previous studies.Seasonal influenza is responsible for significant morbidity and mortality worldwide. In the 1990s, it was estimated to kill 36,000 persons annually in the United States alone and 250,000 to 500,000 persons in the developed world, although hospitalization rates and mortality figures varied considerably from season to season based on the circulating strains (19, 20). Influenza A viruses also have the capability to cause a pandemic if they are sufficiently novel. Strains may emerge whole or in part from animal reservoirs and establish long-term (years to decades) zoonotic lineages in humans (23). The most striking example of this phenomenon occurred in 1918, when an avian virus of the H1N1 subtype crossed the species barrier and established related lineages in two mammalian hosts, swine and humans (16). This pandemic is thought to have killed more than 40 million persons worldwide. In 2009, a novel H1N1 influenza virus of swine origin (H1N1 S-OIV) emerged and is now causing the first pandemic the world has seen in more than 40 years (14). Because of the history of pandemic influenza and the current circulation of a novel pandemic strain, there is intense interest and urgency in understanding viral factors that allow expression of disease in humans.One such virulence factor is the influenza A virus protein PB1-F2 (8). This small (87 to 90 amino acids), 11th gene product was discovered in 2001 in a search for CD8⁺ epitopes in alternative reading frames of influenza A virus genes (2). It is encoded in the +1 reading frame of the PB1 gene segment and is translated from an AUG codon downstream of the PB1 start site, probably accessed through leaky ribosomal scanning. It has been shown to contribute to virulence both directly and indirectly, through modulation of responses to bacteria (3, 11). The exact mechanism(s) through which virulence is increased by PB1-F2 expression, however, is not yet understood. Three effects of PB1-F2 expression have been suggested so far. It has been demonstrated to cause cell death in some cell types (2, 5), it has been shown to induce inflammation by recruitment of inflammatory cells in mice (11), and it has been determined to bind PB1 and to increase the activity of the influenza virus polymerase in vitro (10).The function of the PB1-F2 protein in the life cycle of influenza virus is as unclear as its precise role in virulence. Given that almost all avian influenza virus strains express a full-length PB1-F2 protein (27), it is likely to contribute to survival or transmission in the natural avian host. After introduction of viruses into mammalian hosts such as humans or swine, however, the protein often becomes truncated during adaptation, implying that any effects it might induce are not necessary for virus viability and transmission in these hosts. The 1918 H1N1 virus had a full-length PB1-F2 protein, which has been demonstrated to contribute to virulence in mice (3, 11). During the evolution of H1N1 viruses in humans over time, a stop codon at position 58 in the PB1-F2 amino acid sequence appeared around 1950 and has been retained in the human H1N1 lineage since its reemergence in 1977. Similarly, multiple swine lineages of influenza A virus have had truncations appear at different positions, including position 58, such that 25% of swine PB1-F2 sequences in GenBank lack the C-terminal portion of the protein (27). The H3N2 lineage of viruses in humans has retained a full-length PB1-F2 protein since the introduction of a new PB1 gene segment during the 1968 pandemic, although considerable variation in sequence has occurred during evolution since that time. It is tempting to map these differences in PB1-F2 expression onto patterns of human excess mortality over time, since higher mortality was associated with H1N1 epidemics in the 1930s and 1940s than has been seen since and more excess mortality occurred in recent years with H3N2 viruses than with either H1N1 or influenza B viruses (reviewed in reference 12). Differences in primary virulence or the association with bacteria mediated by PB1-F2 expression could be at least partly responsible for these observed epidemiologic trends.A recent paper from Wise et al. has shown that a 12th influenza A virus gene product, N40, is also expressed from the PB1 gene segment (24). A delicate balance between PB1, PB1-F2, and N40 appears to be in place. Polymerase activity measured by an in vitro assay was affected by changes in this balance, suggesting a potential importance for replication. If these differences translate to differences in replication, then this could be a key factor in virulence in the host. However, to this point, most studies have utilized a single laboratory variant of influenza A virus, A/Puerto Rico/8/34 (H1N1; PR8), in a limited set of cell types, in assays performed in vitro. We undertook this study to determine the relevance of potential changes in replication mediated by PB1-F2 expression, utilizing several different epidemiologically important virus strains. We found that the effects on polymerase activity and in vitro replication efficiency were virus and cell type specific and did not mediate changes in viral lung load in animals.     

A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon

Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.