To elucidate the molecular events concerned with the expression and replication of the influenza virus genome, this proposal focuses on the characterization of the structure and multiple functions of the virus-encoded NS1 protein. The NS1 protein of influenza A virus (NS1A protein) is an unique posttranscriptional regulator that has several activities: inhibition of the nuclear export of mRNA; inhibition of pre-mRNA splicing; and blocking activation of the PKR kinase that would lead to the inhibition of translation. These activities are mediated by the NS1A RNA-binding domain (located near the amino terminal end) that binds to poly(A), U6 small nuclear RNA, and double-stranded RNA, respectively. The NS1 protein of influenza B viruses (NS1B protein) also binds to the same three RNAs. Both NMR and X-ray crystallography will be used to characterize the structural features of the interactions between these two NS1 protein RNA-binding domains and their RNA targets. These studies may lead to the design of compounds that will specifically inhibit this binding and hence the replication of all influenza A and B viruses. The carboxyl half of almost all NS1A proteins contains a second functional domain, the effector domain, which is required for the inhibition of the nuclear export of mRNA and pre-mRNA splicing in vivo. To determine the mechanism of action of the NS1A effector domain: (i) an in vivo assay will be developed to identify the specific NS1A amino acid sequence that functions as an independent effector domain, using chimeric proteins containing NS1A protein sequences linked to the Rev protein of HIV-1; (ii) the specific amino acid sequences in the NS1A effector domain that mediate the nuclear retention of the protein will be identified, analogous to the amino acid sequences that mediate determined using NMR and X-ray crystallography; and (iv) the cellular nuclear proteins that interact with the effector domain of the NS1A protein and that are required for its in vivo functions will be identified; and the cellular function(s) carried out by these nuclear protein will be determined. Initial results indicate that the carboxyl region of not only the NS1A protein but also the NS1B protein causes the induction of apoptosis, or programmed cell death. The NS1 amino acid sequence that is required for apoptosis as well as the cellular protein(s) that mediate NS1 protein-mediated apoptosis will be identified. Finally, these results will serve as a guide to identify specific processes that the NS1 protein regulates in influenza A and B virus-infected cells.
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