The herpes simplex virus type 1 immediate early protein ICP27 is a multifunction regulator of viral gene expression. At early times after infection, ICP27 enters the nucleus and brings with it the SR protein specific kinase SRPK1. ICP27 interacts with spliceosomal complexes through its interaction with SR proteins. Aberrant phosphorylation of SR proteins mediated by ICP27's interaction with SRPK1 results in stalled pre- spliceosomal complexes and the inhibition of cellular splicing. ICP27 encounters Aly/REF, an exon junction protein that functions as an RNA export adaptor, and recruits Aly/REF to HSV-1 transcription-replication sites. ICP27 accesses these sites by binding to the CTD of RNAP II and is involved in the efficient recruitment of RNAP II to HSV-1 genome replication sites. ICP27 binds viral RNA and the ICP27-Aly/REF- RNA complexes are directed to TAP/NXF, the cellular mRNA export receptor. ICP27 interacts with TAP/NXF and the RNP complex is exported to the cytoplasm. ICP27 may also be involved in stimulating translation of bound RNAs. In the extension period, we will further define how ICP27 is directed to sites of RNA processing and in particular define if ICP27 interacts predominantly with unphosphorylated or phosphorylated CTD, and if ICP27 recruits 3' processing factors to the elongating RNAP II complex; define whether or not interactions with ICP4 and/or ICP8 are required for or involved in the interaction of ICP27 with the CTD and in ICP27's recruitment of RNAP II to HSV-1 transcription sites; determine if the modification of RNAP II phosphorylation mediated by ICP22 is required for efficient HSV-1 transcription under conditions in which proteasomal degradation is blocked; determine if the modification of RNAP II mediated by ICP22 is beneficial to viral gene expression to maintain sufficient pools of serine-2 phosphorylated RNAP II CTD for elongation of viral transcripts due to proteasomal degradation of stalled or paused complexes. We will also define the control of HSV-1 RNA export by ICP27 and define the regulation of ICP27 import/export by continuing to probe the arginine methylation state of ICP27 and determine if the affinity of ICP27 for its interacting protein partners is enhanced or regulated by R-methylation; determine if phosphorylation of ICP27 regulates its interactions with its protein partners; determine if other cellular export adaptors are involved in HSV-1 RNA export; determine if phosphorylation regulates ICP27 import or dissociation of the RNA cargo in the cytoplasm; and determine if ICP27 bound to RNA in the cytoplasm stimulates translation. These studies address the mechanisms of action of an essential multifunctional regulator of herpesvirus gene expression, which will help us to elucidate the complex network of virus-cell interactions, 'and which can eventually lead to improved targeted therapeutics and antiviral interventions.
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