Biological characterization of RNA polymerase proteins from human 2009 pandemic H1N1 and low pathogenic avian H5N2 and H9N2 influenza viruses
Myaing, Myint Zu
Date of Issue2016-12-19
School of Biological Sciences
The influenza A genome replication and gene transcription are mediated by polymerase complex composed of three polymerase subunits PA, PB1 and PB2. This polymerase complex is also essential for host adaptation and pathogenesis. To characterize the polymerase complex of 2009 pandemic H1N1 (pH1N1) and low pathogenic avian (LPAI) H5N2 influenza viruses, monoclonal antibodies (mAbs) against these complexes are essential. Therefore, we have challenged mice with purified soluble PA protein (pH1N1/471 and H5N2/F118 strains), insoluble PB2 and PB1 proteins (pH1N1/471 strain). Then, mAbs directed against PA subunit of both strains and PB2 subunit of 2009 pH1N1 strain were successfully generated, while mAb against PB1 subunit was not successful at fusion stage for two attempts. Interestingly, two mAbs-PA(9F5 and 2E2) against pH1N1/471 strain recognized PA protein in virus-infected cells with different staining patterns, which may be explained by their different epitope recognition. In addition, smaller protein products (PA*) were identified by 9F5 in recombinant protein expression, virus-infected cells and mature virus particles. We proved the PA* are PA related and demonstrated the first evidence of their association with the RNP complex. Similar to PA*, PB2* was also revealed by mAb-PB2(4G3), but in the virions, suggesting that PB2* was not virus-associated. However, significance of PB2* remained unclear and it needs further investigation. For the functional analysis of viral polymerase complex, NP and three polymerase subunits of four influenza virus strains have been successfully cloned using a mammalian expression system. H1N1/WSN polymerase complex was found to be the most active than polymerase complexes of other strains tested. In our gene reassortment study, a single reassortant bearing a human-origin PA or PB2 subunit against an avian polymerase background could overcome the host range barrier of avian polymerase in human 293T cells, indicating the PA or PB2 as a major determinant of species tropism and pathogenicity. Again in our RNAi study, potent in vitro inhibition of virus replication was achieved with eight siRNAs against NP and polymerase genes of LPAI H5N2 virus as observed by reduction in mRNA levels, protein expressions and virus titers.