Potential genetic polymorphism of PB2-701 and 702: implication in virus-host interaction of influenza A virus

Abstract

BACKGROUND: PB2-701 of influenza A virus is known as a genetic marker for host adaptation. A D701N mutation of an avian influenza virus can enhance the polymerase activity and viral replication in mammalian cells. In addition, PB2-702 shows host specificity, with most human viruses carry an arginine and most avian viruses carry a lysine at this residue. However, limited polymorphisms at these two residues are found in the natural isolates, limiting the study of their role in the polymerase. METHOD: In order to further elucidate the role of PB2-701/702 in viral fitness and host adaptation, we aim to investigate the potential genetic polymorphism of the PB2-701 and 702 residues by site-directed random mutagenesis of the PB2 gene of the influenza virus in mammalian and avian cells. The polymerase activity, viral replication and pathogenicity of the mutant viruses generated were characterized. RESULTS: A wide range of mutant viruses with different PB2-701 and 702 mutations were successfully isolated, showing that viable viruses with polymorphisms other than PB2-701D/N and 702K/R could be generated. These mutant viruses showed variable polymerase activity in mammalian and avian cells. Several mutants showed enhanced polymerase activity in mammalian cells and comparable viral replication and pathogenicity when compared to the wild-type virus. The variation in the polymerase activity in mammalian cells may be due to the change in net surface charge of the loop around residues 700 – 703 of the PB2 C-terminal when mutations at PB2-701/702 occur. The polymerase activity in mammalian cells generally increases as the surface of the PB2-700 – 703 region becomes more positively charged. On the other hand, some PB2-701/702 mutants (e.g. 701A/702E and 701S/702F) showed reduced polymerase activity and viral replication in mammalian cells. One of them (701A/702E) also showed lower pathogenicity in mice. Importin-α4 was found to have a role in the reduction of the polymerase activity and viral replication of these mutants. Knocking-down the importin-α4 of the mammalian cells enhanced the polymerase activity and/or viral replication of these mutant viruses at 37°C, while the importin-α4 inhibitory effect is not significant in other mutant viruses with high polymerase activity. CONCLUSION: This study demonstrated the potential genetic polymorphism at the residues 701 and 702 of PB2. Some mutant viruses have enhanced polymerase activity when compared to the wild-type, while some have reduced polymerase activity, viral replication and pathogenicity. We also demonstrated that importin-α4 may be responsible for the inhibitory effect observed. Overall, this study may shed light on the study of the role of PB2-701/702 in virus-host interaction.