Influenza virus protein PB1-F2 inhibits the induction of type I interferon by binding to MAVS and decreasing mitochondrial membrane potential.

PB1-F2 is a small, 87- to 90-amino-acid-long protein encoded by the +1 alternate open reading frame of the PB1 gene of most influenza A virus strains. It has been shown to contribute to viral pathogenicity in a host- and strain-dependent manner, and we have previously discovered that a serine at position 66 (66S) in the PB1-F2 protein increases virulence of the 1918 and H5N1 pandemic viruses. Recently, we have shown that PB1-F2 inhibits the induction of type I interferon (IFN) at the level of the MAVS adaptor protein. However, the molecular mechanism for the IFN antagonist function of PB1-F2 has remained unclear. In the present study, we demonstrated that the C-terminal portion of the PB1-F2 protein binds to MAVS in a region that contains the transmembrane domain. Strikingly, PB1-F2 66S was observed to bind to MAVS more efficiently than PB1-F2 66N. We also tested the effect of PB1-F2 on the IFN antagonist functions of the polymerase proteins PB1, PB2, and PA and observed enhanced IFN inhibition by the PB1 and PB2 proteins in combination with PB1-F2 but not by the PA protein. Using a flow cytometry-based assay, we demonstrate that the PB1-F2 protein inhibits MAVS-mediated IFN synthesis by decreasing the mitochondrial membrane potential (MMP). Interestingly, PB1-F2 66S affected the MMP more efficiently than wild-type PB1-F2. In summary, the results of our study identify the molecular mechanism by which the influenza virus PB1-F2 N66S protein increases virulence.

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-ß 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-ß 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 N66S mutation in the PB1-F2 protein of influenza A virus increases virulence by inhibiting the early interferon response in vivo.

The PB1-F2 protein of influenza A virus can contribute to viral pathogenesis of influenza virus strains. Of note, an N66S amino acid mutation in PB1-F2 has been shown to increase the pathogenesis associated with H5N1 Hong Kong/1997 and H1N1 Brevig Mission/1918 influenza viruses. To identify the mechanism of enhanced immunopathology, we evaluated the host response to two isogenic viruses that differ by a single amino acid at position 66 of the PB1-F2 protein. Various components of the adaptive immune response were ruled out as factors contributing to pathogenesis through knockout mouse studies. Transcriptional profiling of lungs from PB1-F2 66S-infected mice revealed an early delay in innate immune responses. In particular, enhanced activation of type I interferon (IFN) pathway genes, including IFN-ß, RIG-I, and numerous interferon-inducible genes, was not observed until day 3 postinfection. The N66S mutant virus caused increased cellularity in the lungs, as a result of monocyte and neutrophil infiltration. Furthermore, numerous cytokines and chemokines related to monocyte and neutrophil migration and maturation were upregulated. The cellular infiltration and increased cytokine expression corresponded to increased PB1-F2 66S titer. These data suggest that PB1-F2 N66S may contribute to the delay of innate immune responses, allowing for unchecked viral growth and ultimately severe immunopathology observed in the lungs.

PB1-F2 expression by the 2009 pandemic H1N1 influenza virus has minimal impact on virulence in animal models.

Unlike previous pandemic viruses, the 2009 H1N1 pandemic influenza virus does not code for the virulence factor PB1-F2. The genome of the 2009 H1N1 virus contains three stop codons preventing PB1-F2 expression; however, PB1-F2 production could occur following genetic mutation or reassortment. Thus, it is of great interest to understand the impact that expression of the PB1-F2 protein might have in the context of the 2009 pandemic influenza virus, A/California/04/2009 (Cal/09). We have addressed this question by generating two Cal/09 viruses with productive PB1-F2 open reading frames containing either an asparagine at position 66 of PB1-F2 (66N) or a serine at position 66 (66S): this N66S change has previously been shown to be associated with increased virulence in mice. We used these viruses to investigate the effect on virulence conferred by expression of the 66N or the 66S PB1-F2 protein in both in vitro and in vivo systems. Our results show enhanced replication of the 66S virus in A549 cells, while studies of BALB/c and DBA/2 mice and ferrets revealed no significant differences in symptoms of infection with wild-type Cal/09 versus the 66N or 66S virus variant. Also, coinfection of mice with Streptococcus pneumoniae and the different viruses (recombinant wild-type [rWT] Cal/09 and the 66N and 66S viruses) did not result in significant differences in mortality. Mice infected with either PB1-F2-expressing virus did demonstrate altered protein levels of proinflammatory cytokines; differences were observed to be greater in infection caused by the 66S virus. In summary, our study demonstrates that PB1-F2 expression by the Cal/09 virus modulates the immune response to infection while having a minimal effect on virus virulence in two mammalian models.

The influenza A virus protein PB1-F2: killing two birds with one stone?

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. The PB1-F2 protein has been shown to contribute to viral pathogenicity, but the molecular mechanisms for mediating virulence have been unclear. Previous reports demonstrate that PB1-F2 promotes cell death, causes immunopathology and increases pro-inflammatory responses. Our group has identified a single point mutation from asparagine (N) to serine (S) at position 66 in the PB1-F2 protein that dramatically increases the virulence of highly pathogenic avian H5N1 influenza viruses and of the 1918 pandemic strain. In search for the mechanism by which PB1-F2 N66S increases pathogenicity, we have identified and characterized a novel function of PB1-F2, i.e. interferon antagonism, both in vitro and in the mouse model. Here, we discuss a hypothesis for a possible molecular link between the pro-apoptotic and anti-interferon functions of PB1-F2.