The long-term goal of this study is to understand the assembly of influenza viruses at the molecular level. Although the replication cycle of influenza virus has been defined in general terms, the molecular mechanisms by which viral and cellular molecules contribute to the different steps of viral assembly are largely unknown. Previous attempts to address this issue relied mainly on virus-infected cells because of the lack of efficient systems for reconstituting the various steps of the assembly process. The applicant has now established the systems needed to generate influenza virus-like particles and infectious influenza viruses entirely from cloned cDNA, and the tools are at hand to answer previously elusive questions in this important area of research.
Aim 1. To elucidate the mechanisms by which M1 and NS2 function in RNP export from the nucleus. After transcription and replication of viral RNA in the nucleus, the newly synthesized viral RNA, nucleoprotein (NP) and polymerase proteins form the ribonucleoprotein complex (RNP), which is then exported to the cytoplasm. By exploiting capabilities to reconstitute influenza virus assembly, it may be possible to determine the exact role(s) of the M1 and NS2 proteins in RNP nuclear export.
Aim 2. To determine the importance of NP actin-binding activity for RNP association with the cytoskeleton and in the viral replication cycle. The hypothesis predicting that RNP associated with the cytoskeleton after export from the nucleus, through actin-binding activity of NP, will be tested using a new reverse genetics system.
Aim 3. To understand the role of M2 protein in virion formation. Prior observations demonstrating a role of the ectodomain in M2 virion incorporation and a role of the cytoplasmic tail in viral replication underpin studies to test the contributions of these domains in particle formation.
Aim 4. To determine the role of the NA protein and its RNA segment in particle formation. Observations that influenza viruses capable of growing without viral sialidase still maintain a truncated NA gene have prompted studies to determine the requirement for the NA protein and its RNA segment in efficient virion formation.
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