The herpes simplex virus 1 (HSV-1) multifunctional protein lCP27 is essential for viral replication. It is required for appropriate expression of viral early and late proteins and contributes to host shut-off. While it has been shown that ICP27 affects transcription, we have focused on its post-transcriptional activities. We identified the stages in RNA processing where ICP27 acts and characterized several crucial intermolecular interactions. ICP27 inhibits host cell splicing, which contributes to the shut-off of host protein synthesis and we defined the stage of splicing that is inhibited and showed that it interacts with and alters essential splicing factors required for splicesome assembly. Because spliced messages are more efficiently targeted for export than transcripts that do not interact with the spliceosome, splicing inhibition by ICP27 also results in the alteration of cellular export pathways. We showed that ICP27 binds to viral intronless RNAs and found that ICP27 interacts with a cellular export factor, Aly/REF that guides spliced transcripts to a major export pathway. By recruiting Aly/REF, ICP27 enables HSV-1 intronless RNAs to be efficiently exported. In this proposal, we will focus on determining how this multifunctional protein is regulated to perform its various activities during viral infection.
The specific aims are: 1) To define how ICP27 is directed to sites of RNA processing by determining if ICP27 associates with RNA polymerase II (RNAP II). We will determine if ICP27 binds, directly or indirectly to the C-terminal domain (CTD) and if it associates with the initiating or elongating complex; define the region of ICP27 required for interaction with RNAP II; determine if ICP27 binds to the processing factors with which it interacts as part of the RNAP II complex and if ICP27 recruits factors to RNAP II, and determine if ICP27 helps to recruit RNAP Il to HSV-1 replication sites. 2) To define the control of HSV-1 RNA export by ICP27 and to define the regulation of ICP27 import/export. We will probe the arginine methylation state of the RGG box of ICP27 and determine if the affinity of ICP27 for Aly/REF or its RNA cargo is enhanced by R-methylation; determine if phosphorylation of ICP27 by SRPK1 or CK2 unmasks the Aly/REF binding site or the NES; determine the RNA sequence(s) or structures required for ICP27 recognition and define the requirement of RNA binding for ICP27 export, and determine what modifications to ICP27 regulate its import and export cycle.
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