Study the Pa Subunit of Influenza a Virus Polymerase for the Regulation of Virus Replication and Pathogenicity

Influenza A viruses are causative agents of contagious respiratory disease in human. The pandemic strains could be highly pathogenic and result in death in infected humans. The viral RNA-dependent RNA polymerase, which is comprised of three subunits (PB1, PB2 and PA), functions as a transcriptase and a replicase for the virus genome, virion RNA (vRNA). For transcribing viral mRNA, the viral polymerase excises capped RNA primer from cellular pre-mRNA by the intrinsic endonuclease activity. In contrast, the viral RNA polymerase copies vRNA to complementary RNA (cRNA) or cRNA to vRNA without a primer in presence of viral nucleocapsid protein (NP). Since the viral polymerase controls the replication of virus genome, it has been considered as a virulence factor for influenza virus infection. From our previous observation, the recombinant virus that contains the internal genes from highly pathogenic strain, A/HK/483/97/H5N1 (HK483), replicates 100-fold faster than the chimeric virus containing polymerase and NP genes from a low pathogenic strain, A/Udorn/1972/H3N2 (Ud). After comparing the activities of HK483 and Ud polymerases by using a transfection-reporter system, we found that the polymerase of the highly pathogenic influenza A virus, HK483, catalyzes viral RNA synthesis more efficiently. Furthermore, a serial comparison for different polymerase subunit combinations has demonstrated that the PA subunit may play an important role for this phenomenon. Our first goal in this grant proposal is to determine the residues on the PA subunit that enhance the activities of influenza polymerase. In order to identify the specific catalytic step(s) and to elucidate the possible mechanisms that may be involved in this enhancement, we will establish an in vitro reconstitution system with purified polymerase complexes and a synthetic vRNA template for the activities. This assay system will be also applied to compare the activities of the polymerases from the pandemic strains. Since several evidences have implicated that host or viral factors participate in influenza virus RNA synthesis, we will use the affinity purification to identify the specific host or viral proteins that interact with the specific influenza polymerase complex from highly pathogenic or low pathogenic virus. In addition, the identified interacting proteins will be tested in the established in vitro assays to elucidate the possible mechanism. The whole study may provide the basis for the development of anti-viral drugs.

Project ID:PC10001-1164
External Project ID:NSC100-2320-B182-001