Modulation and Utilization of RNA Polymerase III by Herpes Simplex Virus

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The genome of herpes simplex virus encodes mRNAs for about 90 protein coding genes in addition to numerous noncoding RNAs. Sets of these genes are transcribed in a sequential manner by RNA pol II. The timing of their expression is dependent on the cis-acting sites for cellular transcription factors in their promoters, their requirements for virus transcription factors, and the state of viral DNA replication. Multiple viral proteins modify or sequester elements of the pol II transcription machinery to enable the robust transcription of the viral genome, which consequently attenuates cellular mRNA transcription. We have found that RNA pol III and some of its transcription factors also bind to the HSV genome. RNA pol III transcribes tRNAs and other cellular non-coding RNAs. We also show that aspects of the binding of pol III to the viral genome are similar to those seen for pol II with respect to the requirement for viral activators and the effect of viral DNA replication. However, unlike with pol II, we find that the transcription of specific cellular pol III transcription units, namely tRNA genes, is increased by infection. We hypothesize that RNA pol III is functioning on the HSV genome to transcribe noncoding RNAs, and that the function of pol III on the genome is regulated by some of the same cis- and trans- acting elements that regulate pol II transcription of the genome. We further hypothesize that HSV infection induces a specific type of pol III transcription by altering the abundance or activity of the pol III transcription factors that bind to cellular pol III promoters. Two specific aims are proposed to address these hypotheses. We will determine the basis for the association of pol III with the HSV genome and the resulting pol III transcripts, and determine how HSV affects cellular pol III transcription. Other DNA viruses encode pol III transcripts; however, this is the first study to show that pol III associates with the HSV genome. It may be that known HSV noncoding RNAs, or yet to be discovered noncoding RNAs, are transcribed by pol III. Other viruses are also known to stimulate pol III transcription. The activation of pol III transcription by HSV may promote the pol III transcription of the viral genome as it does for adenovirus or EBV, and/or provide for the optimal levels of tRNAs for the translation of viral protein coding mRNAs. This exploratory grant will determine the viral and cellular RNA pol III transcriptome of infected cells, and the mechanisms by which virus infection modulates pol III transcription as prelude to a more in depth investigation of the interaction of pol III with HSV and its significance for viral infection.