Herpes simplex virus type-1 (HSV-1) causes changes in host cell transcriptional programs during the hours after it infects mammalian cells. The transcription of protein-encoding genes by RNA polymerase II (Pol II) is largely repressed after HSV-1 infection, however the mechanism of this repression is not fully understood. It is hypothesized that specific non-coding RNAs (ncRNAs) that are upregulated during viral infection act as global, transacting repressors of Pol II transcription after HSV-1 infection. The specific ncRNAs proposed to act as repressors are mouse B2 RNA and human Alu RNA, which are transcribed by RNA polymerase III from short interspersed elements (SINEs) that litter the mouse and human genomes, respectively. This hypothesis is based on prior studies investigating the global repression of mRNA transcription that occurs during heat shock;B2 and Alu RNAs were found to directly repress Pol II transcription after heat shock in mouse and human cells, respectively. The pilot study described here seeks to determine whether B2 and Alu RNAs function in the repression of Pol II transcription after HSV-1 infection. The proposal has three highly focused Specific Aims. 1) Identify the Pol II-transcribed host cell genes that are repressed after HSV-1 infection of mouse and human cells by measuring changes in Pol II occupancy across the genomes before and after viral infection. 2) Determine whether B2 RNA and Alu RNA contribute to repression of host cell Pol II transcription after HSV-1 infection by knocking down the ncRNAs and determining the effect on Pol II occupancy across the genome before and after infection. 3) Determine whether B2 and Alu RNAs associate with Pol II and with the promoters of repressed genes in response to HSV-1 infection using two established cell-based assays. The proposed research will determine whether B2 and Alu RNAs mediate HSV-1 induced repression of host cell transcription. In testing this hypothesis, a genome-wide understanding of how HSV-1 infection reprograms Pol II occupancy on mRNA genes will be obtained, as well as an understanding of how HSV-1 affects the activity of B2 RNA and Alu RNA. The results of these pilot studies will enable a new line of research on the interplay between HSV-1 infection, SINE ncRNA transcription and function, host cell transcription, and transcription of viral genes, which will be the focus of a future R01 grant application. Lastly, since B2 RNA and Alu RNA levels are known to increase after infection by other viruses and in response to other biological processes, our studies could provide general insight into how these ncRNAs control transcription in multiple situations.
Properly controlling gene expression is essential to sustaining life and avoiding many diseases and cancers;mRNA transcription by RNA polymerase II is central to this process. The proposed studies will test a new hypothesis for how host cell mRNA transcription is repressed when Herpes Simplex Virus type-1 infects human and mouse cells. This research will be an important first step in understanding how the virus orchestrates major changes in transcriptional programs during infection.