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

Abstract

Introduction: PB2-701 of influenza A virus is a genetic marker for host adaptation. A D701N mutation of an avian virus can enhance the polymerase activity and viral replication in mammalian cells. PB2-702 shows host specificity, with most human viruses carry an arginine and most avian viruses carry a lysine. However, limited polymorphisms at these two residues are found in the natural isolates, limiting the study of their role in the polymerase. Objectives: 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 virus. Methods: To elucidate the role of PB2-701/702 in viral fitness, site-directed random mutagenesis of the PB2 gene was performed to generate recombinant viruses with random mutations at PB2-701/702 in mammalian and avian cells. The polymerase activity, viral replication and pathogenicity of the mutant viruses generated were characterized. Results: A wide range of viruses with different PB2-701/702 mutations were isolated. Several mutants demonstrated enhanced polymerase activity in mammalian cells and comparable viral replication and pathogenicity in mice when compared to the wild-type virus. Surface electrostatic charge prediction on the PB2 structural model revealed that 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 mutants had reduced polymerase activity and viral replication in mammalian cells. One of them (701A/702E) also had lower pathogenicity in mice. Distinct tissue tropism of the PB2-701/702 mutants was observed in chicken embryos. It was found that importin-α4 has a role in the reduction of the polymerase activity in some mutants. Knocking-down the importin-α4 of the mammalian cells enhanced the polymerase activity of the mutants with low polymerase activity, while the importin-α4 inhibitory effect is not significant in other mutants with high polymerase activity. Conclusion: Overall, this study demonstrated the potential genetic polymorphisms at PB2-701/702 and revealed the potential role of importin-α4 in modulating the polymerase activity. The findings in this study may lead to the further study of the role of PB2-701/702 in virus-host interaction and contribute to the control of influenza.