Transcription and replication of influenza viral genomic RNAs are mediated by a unique virus coded polymerase that consists of three polypeptide subunits, namely, PB1, PB2 and PA. The synthetic steps catalyzed by viral polymerase, which are also influenced by other viral and cellular proteins, are not well understood. Temperature sensitive (ts) mutants of influenza virus that are defective in transcription and replication will be systematically analyzed for specific defects in nuclear transport, polymerase complex formation, polymerase RNP complex formation and catalytic function using the recently developed reagents and methodologies. The mechanism of anti-termination mediated by nucleoprotein (NP) in the synthesis of full length cRNA templates will be examined to determine if free uncomplexed NP binds to nascent cRNA strands to facilitate this step. In vitro translated NP and polymerase proteins will be used in vitro reactions to determine if free NP alone can cause antitermination and if individual P proteins can complement ts functions in vitro. This study will provide us an unique opportunity to understand how the viral polymerase subunits interact with each other and with other viral components to perform many complex functions in an infected cell. Since influenza virus transcription/replication differs markedly from non-segmented negative strand viruses like VSV, detailed study of its replication may reveal many yet unidentified strategies employed by viruses in general, and will provide valuable comparative data.
