The direct infection of humans with highly pathogenic avian H5N1 influenza viruses has suggested viral mutation as one mechanism for the emergence of novel human influenza A viruses. Although the polymerase complex is known to be a key component in host adaptation, mutations that enhance the polymerase activity of avian viruses in mammalian hosts are not fully characterized. The genomic comparison of influenza A virus isolates has identified highly conserved residues in influenza proteins that are specific to either human or avian viruses, including 10 residues in PB2. We characterized the activity of avian polymerase complexes containing avian-to-human mutations at these conserved PB2 residues and found that, in addition to the E627K mutation, the PB2 mutation T271A enhances polymerase activity in human cells. We confirmed the effects of the T271A mutation using recombinant WSN viruses containing avian NP and polymerase genes with wild-type (WT) or mutant PB2. The 271A virus showed enhanced growth compared to that of the WT in mammalian cells in vitro. The 271A mutant did not increase viral pathogenicity significantly in mice compared to that of the 627K mutant, but it did enhance the lung virus titer. Also, cell infiltration was more evident in lungs of 271A-infected mice than in those of the WT. Interestingly, the avian-derived PB2 of the 2009 pandemic H1N1 influenza virus has 271A. The characterization of the polymerase activity of A/California/04/2009 (H1N1) and corresponding PB2 mutants indicates that the high polymerase activity of the pandemic strain in mammalian cells is, in part, dependent on 271A. Our results clearly indicate the contribution of PB2 amino acid 271 to enhanced polymerase activity and viral growth in mammalian hosts.

Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA's reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix ␣4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis.

Viruses require the host translational apparatus to synthesize viral proteins. Host stress response mechanisms that suppress translation, therefore, represent a significant obstacle that viruses must overcome. Here, we report a strategy whereby the mammalian orthoreoviruses compartmentalize the translational machinery within virus-induced inclusions known as viral factories (VF). VF are the sites of reovirus replication and assembly but were thought not to contain ribosomes. It was assumed viral mRNAs exited the VF to undergo translation by the cellular machinery, and proteins reentered the factory to participate in assembly. Here, we used ribopuromycylation to visualize active translation in infected cells. These studies revealed that active translation occurs within VF and that ribosomal subunits and proteins required for translation initiation, elongation, termination, and recycling localize to the factory. Interestingly, we observed components of the 43S preinitiation complex (PIC) concentrating primarily at factory margins, suggesting a spatial and/or dynamic organization of translation within the VF. Similarly, the viral single-stranded RNA binding protein σNS localized to the factory margins and had a tubulovesicular staining pattern that extended a short distance from the margins of the factories and colocalized with endoplasmic reticulum (ER) markers. Consistent with these colocalization studies, σNS was found to associate with both eukaryotic translation initiation factor 3 subunit A (eIF3A) and the ribosomal subunit pS6R. Together, these findings indicate that σNS functions to recruit 43S PIC machinery to the primary site of viral translation within the viral factory. Pathogen-mediated compartmentalization of the translational apparatus provides a novel mechanism by which viruses might avoid host translational suppression.

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.

In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains. This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet, despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/California/04/09 (ACal-04/09), to be predominantly spherical by immunufluorescence and electron microscopy analysis in human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain. Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1), a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between influenza morphology and efficient cell-to-cell spread in vitro. Furthermore, we observed efficient virus-like particle production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of spherical progeny, which may contribute to the rapid spread of the pandemic virus.