We intend to determine the mechanism of the transcription and replication of the influenza virus genome and the roles of virus- specific proteins in these processes. To answer unresolved questions about the roles of the viral P(PB1, PB2 and PA) proteins in capped RNA-primed viral mRNA synthesis, we will: (i) determine whether the P protein complexes (containing the three P proteins) that are not associated with viral nucleocapsids in the infected cell function in the initiation and reinitiation of viral mRNA synthesis; and (ii) using the large amounts of the P proteins that we have expressed with baculovirus vectors, reconstitute P protein complexes that are active in mRNA synthesis. We will also use baculovirus expressed nucleocapsid (NP) protein to reconstitute nucleocapsids active in mRNA synthesis, thereby enabling us to identify the sequences in virion RNA (vRNA) that are needed for the initiation and termination of mRNA synthesis. The interferon-induced Mx protein specifically inhibits influenza viral mRNA synthesis in the infected cell. We will determine whether baculovirus expressed Mx protein inhibits specific step(s) of viral mRNA synthesis in vitro and, if so, will establish whether this inhibition is mediated by a specific interaction of the Mx protein with one more P proteins. These studies could lead to the development of antivirals directed against all influenza virus strains. Using infected cell nuclear extracts that catalyze the synthesis of the templates for vRNA synthesis (full-length copies of the vRNAs), we have shown that the NP protein is required for antitermination at the poly (A) site used during mRNA synthesis. We will determine whether other viral proteins are also needed for antitermination and whether the NP protein forms nucleocapsids with the newly synthesized template RNAs. We will also identify the viral proteins (e.g., P proteins, the two nonstructural proteins NS1 and NS2) that participate in the unprimed initiation of template RNA into vRNA, we intend to identify the viral proteins that are needed for the unprimed initiation and elongation of vRNA chains. The splicing of the viral NS1 and M1 mRNAs by host nuclear enzymes is controlled in infected cells such that the steady-state amount of the spliced mRNAs is only about 5-10% of that of the unspliced mRNAs. We have showned that the NS1 and M1 mRNAS are poor substrates for in vitro splicing. We intend to determine: (i) the reason for the inability of these two mRNAs to function efficiently in splicing; and (ii) whether splicing in the infected cell is controlled solely by the structure of the NS1 and M1 mRNAs, or requires the participation of a viral protein(s) that activates the splicing of these two mRNAs.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Method to Extend Research in Time (MERIT) Award (R37)
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Experimental Virology Study Section (EVR)
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Sloan-Kettering Institute for Cancer Research
New York
United States
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Kuo, Rei-Lin; Li, Li-Hsin; Lin, Sue-Jane et al. (2016) Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation. J Virol 90:4696-4705
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