Abstract
Influenza A viruses encode several accessory proteins that have host- and strain-specific effects on virulence and replication. The accessory protein PA-X is expressed due to a ribosomal frameshift during translation of the PA gene. Depending on the particular combination of virus strain and host species, PA-X has been described as either acting to reduce or increase virulence and/or virus replication. In this study, we set out to investigate the role PA-X plays in H9N2 avian influenza viruses, focusing on the natural avian host, chickens. We found that the G1 lineage A/chicken/Pakistan/UDL-01/2008 (H9N2) PA-X induced robust host shutoff in both mammalian and avian cells and increased virus replication in mammalian, but not avian cells. We further showed that PA-X affected embryonic lethality in ovo and led to more rapid viral shedding and widespread organ dissemination in vivo in chickens. Overall, we conclude PA-X may act as a virulence factor for H9N2 viruses in chickens, allowing faster replication and wider organ tropism.
Highlights
Influenza A viruses (IAV) have segmented negative-sense RNA genomes encoding 10 core proteins and a variable number of strain-specific accessory proteins [1, 2]
As expected, ablating PA-X expression resulted in loss of host shutoff activity, showing that, as with other IAV strains, the shutoff activity of H9N2 viruses is partly due to PA-X expression
We found that PA-X expression resulted in slightly increased replication in mammalian Madin-Darby canine kidney (MDCK) cells but had no effect on titres in primary chicken cells or eggs, virus with PA-X caused more embryonic lethality in ovo at higher input doses
Summary
Influenza A viruses (IAV) have segmented negative-sense RNA genomes encoding 10 core proteins and a variable number of strain-specific accessory proteins [1, 2]. Due to its small genome size and nuclear replication, IAV has evolved a number of ways to increase its protein coding capacity including the use of splice variants (M2, NEP and the more recently discovered M42, PB2-S 1 and NS3 proteins), encoding multiple open reading frames (ORFs), both nested and overlapping on a single gene segment (e.g. PB1-F 2, PB1- N40, NA43) and ribosomal frameshifts leading to multi-ORF fusion proteins (PA-X) [3,4,5,6,7,8]. PA is an integral part of the influenza virus RNA-dependent RNA polymerase (RdRp) and contains two functional domains, an N-terminal endonuclease (endo) domain, responsible for cleaving host capped RNAs used to prime viral transcription, and a C-terminal domain, associated with the core of the RdRp [9]. PA-X has been shown to mediate degradation of host cell mRNAs and disruption of host mRNA processing, leading to host cell shutoff and a dampened innate immune response [11,12,13,14,15,16,17]
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