Abstract
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.
Highlights
Influenza viruses cause annual epidemics and occasional pandemics which may result in up to 50 million excess deaths as seen during the outbreak in 1918 [1]
We have previously established that a single amino acid substitution (N66S) in the viral PB1-F2 protein causes increased virulence in an H5N1 and the 1918 pandemic virus
We demonstrate that the inhibition of IFN by PB1-F2 N66S occurs at the level of the mitochondrial antiviral signaling protein (MAVS), a key player in the IFN production pathway
Summary
Influenza viruses cause annual epidemics and occasional pandemics which may result in up to 50 million excess deaths as seen during the outbreak in 1918 [1]. The virus strains causing the influenza pandemics that occurred in the years of 1918, 1957 and 1968, all have been found to express the virulence factor PB1-F2. A recent study has identified a single point mutation at amino acid position 66 in the PB1-F2 protein of the 1918 pandemic and of an H5N1 influenza virus strain, which is associated with increased virulence [4]. This mutation consists of a change of the amino acid asparagine (66N) to serine (66S) and causes increased weight loss and viral loads in infected mice. We aim to verify these findings in vitro and elucidate the molecular mechanism for the IFN antagonism function of PB1-F2 N66S
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