Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is a multifunctional regulatory protein that is essential for viral lytic infection. ICP27 contributes to the shut off of host protein synthesis and is required for the appropriate expression of viral early and late gene products. While ICP27 has been shown to affect the transcription of late genes, it acts predominantly at the post-transcriptional level affecting RNA processing and export. ICP27 performs its activities by interacting with RNA and with a myriad of proteins. ICP27 binds viral RNAs to facilitate their export. Further, ICP27 interacts with itself to form multimers, and with HSV-1 proteins ICP4 and ICP8, as well as with cellular proteins including: SR splicing proteins, SR protein kinase 1, spliceosomal protein SAP145, hnRNP K, CK2, RNA export protein Aly/REF, mRNA export receptor TAP/NXF1 and RNA polymerase II. The regions of ICP27 involved in these interactions have been broadly mapped to protein motifs in both the N-terminal and C-terminal halves of the molecule. Although several protein motifs were identified based upon sequence comparisons, no structural information on ICP27 has been generated. The goals of this project are to elucidate the structure of the functional domains of ICP27 and to chart the complete array of its dynamic interactions during viral infection.
The specific aims are: 1) To perform a structural analysis of the functional domains of ICP27 by probing the solution structure of the N-terminal leucine-rich NES-like sequence, the highly acidic region, the NLS, the RGG box RNA-binding domain and R2, a second arginine-rich region; and the C-terminal KH RNA binding domains and zinc-finger domain by nuclear magnetic resonance; 2) to create mutations in critical residues that will inactivate the structure of each domain and to test those mutations for their effects on the activities and interactions of ICP27 during infection; and 3) to chart the dynamics of the interactions of ICP27 during viral infection with wild type and mutant viruses bearing targeted structural motif mutations using fluorescence microscopy strategies to view the interactions of ICP27 with cellular and viral factors throughout the infectious cycle in living cells; and to elucidate the full array of ICP27 interacting partners and its participation in different protein complexes by purifying and analyzing these complexes biochemically and by Mass Spectrometry.
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