The infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in dramatic alterations to the host cell nucleus, so that viral genes are expressed at high levels while cellular genes are nearly completely suppressed. These changes are mediated by a small number of viral regulatory proteins that are amenable to biochemical and genetic analysis. This proposal focuses on ICP27, one of the key HSV-1 regulators. ICP27 is an unusual regulator that modulates genes post-transcriptionally, affecting multiple aspects of pre-mRNA metabolism. However, the specific molecular mechanisms used by ICP27 are unknown. In this application, genetic, biochemical and cell biological approaches are proposed to elucidate these mechanisms.
The first aim i s to characterize ICP27 by engineering and analyzing novel HSV-1 mutants. A comprehensive set of mutants that contain in-frame deletions in the ICP27 gene will be constructed and characterized. In addition, a recently isolated viral revertant, M16R, will be analyzed to understand how a truncated amino-terminal form of ICP27 can carry out essential replicative activities.
The second aim i s to study ICP27's recently discovered RNA-binding activity. In vitro RNA-binding assays and selection-amplification technology will be used to identify specific RNA sequences or structures which are recognized by ICP27. Additional experiments will test the hypothesis that post-translational methylation of ICP27 alters its interaction with RNA.
The third aim i s to understand the biological relevance of ICP27's nuclear shuttling activity. Viral ICP27 mutants will be surveyed to see which are defective in shuttling. Experiments will also be carried out to determine whether ICP27's nuclear export is an active, energy-dependent process. If so, ICP27's nuclear export signal will be mapped.
The final aim i s to test whether ICP27 is a virus-encoded hnRNP protein, a hypothesis which could explain ICP27's diverse effects on gene expression. To address this, the protein components of infected cell-hnRNP complexes will be characterized. The research in this proposal will increase our understanding of the basic replication pathways of herpesviruses, and will provide insight into how specific trans-acting factors can regulate genes post-transcriptionally.
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