The herpes simplex virus 1 (HSV-1) immediate early regulatory protein ICP27 is essential for virus replication. This 63 kilodalton protein is required for late gene expression, sufficient levels of some early gene products and efficient DNA synthesis. ICP27 also contributes to the shut off of host protein synthesis. While it has been shown that ICP27 affects transcription, studies in Dr. Sandri-Goldin's laboratory have demonstrated that ICP27 also acts post-transcriptionally at the level of RNA processing. Thus, stimulation of late gene expression by ICP27 is accomplished in part by increased 3' processing at specific polyadenylation sites. ICP27 contributes to host shut off by impairing splicing thus decreasing levels of spliced host mRNAs. Furthermore, recent studies suggest that ICP27 plays a role in nuclear export of HSV-1 mRNA. The goal of this proposal is to elucidate the mechanisms by which ICP27 affects RNA processing.
The specific aims are: 1) To define how ICP27 enhances poly(A) site selection by: characterizing responsive sits during HSV-1 infection; determining the RNA sequence requirements for ICP27 binding and identifying regions of ICP27 involved in RNA binding. and determining how RNA binding enhances poly(A) site recognition by probing ICP27 interactions with polyadenylation factors. 2) To define how ICP27 interferes with host cell splicing by: determining the stage(s) in the splicing reaction affected by ICP27; identifying proteins with which ICP27 interacts and determining if there are changes in their phosphorylation, and determining if ICP27 binds snRNA. 3) To define the role of ICP27 in RNA export by: determining which RNAs are bound to ICP27 by UV cross-linking studies; ascertaining the importance ICP27 to the export of HSV-1 mRNAs by nuclear/cytoplasmic fractionation studies and by in situ hybridization/immunofluorescence analysis, and finally, identifying proteins that interact with the nuclear export signal of ICP27 using the yeast two-hybrid system.
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