Characterization of Influenza A Viruses with Polymorphism in PB2 Residues 701 and 702

Abstract

Introduction and Project Objectives: The 701 and 702 positions of influenza PB2 polymerase subunit are previously shown to have roles in host range. However, limited polymorphisms at these two residues are identified in natural isolates, thereby limiting the study of their role in the polymerase. In this study, we aim to investigate the potential genetic polymorphism of the PB2-701 and 702 residues and their roles in viral properties. Methods / Implementation: Site-directed random mutagenesis at residues 701 and 702 of the PB2 gene of the influenza virus in mammalian and avian cells were performed. Mutant viruses were rescued by reverse genetics. The polymerase activity, viral replication and pathogenicity of the mutant viruses generated were characterized. Results / Outcome: Thirty-one viable viruses were generated by random mutagenesis and reverse genetics, indicating that the PB2-701 and 702 positions can tolerate a wide range of amino acids. These mutants demonstrated varying polymerase activities and viral replication rates in mammalian and avian cells. Notably, some mutants displayed enhanced polymerase activity, yet their replication kinetics were comparable to the wild-type virus. Surface electrostatic charge predication on the PB2 structural model revealed that the viral polymerase activity in mammalian cells generally increases as this region becomes more positively charged. One of the mutants (701A/702E) showed much reduced pathogenicity in mice while others had a pathogenicity similar to the wild-type level. Distinct tissue tropisms of the PB2-701 and 702 mutants were observed in infected chicken embryos. Conclusion: Overall, this study demonstrates that the PB2-701 and 702 region has a high degree of sequence plasticity and sequence changes in this region can alter virus phenotypes in vitro and in vivo.