Do the B2 and Alu ncRNAs Repress Host Cell mRNA Transcription During HSV-1 Infect

描述（由申请人提供）：1型单纯疱疹病毒（HSV-1）在感染哺乳动物细胞后数小时内引起宿主细胞转录程序的变化。1型单纯疱疹病毒感染后，RNA聚合酶II （Pol II）对蛋白编码基因的转录受到很大程度的抑制，但这种抑制的机制尚不完全清楚。据推测，在病毒感染期间上调的特异性非编码rna （ncRNAs）在HSV-1感染后作为全局的Pol II转录抑制因子进行交易。被提出作为阻遏物的特异性ncRNAs是小鼠B2 RNA和人类Alu RNA，它们分别由RNA聚合酶III从小鼠和人类基因组中的短穿插元件（short interspersed element, SINEs）转录而成。这一假设是基于先前的研究，研究了在热休克期间发生的mRNA转录的全球抑制；发现B2和Alu rna分别在小鼠和人细胞热休克后直接抑制Pol II转录。本文描述的初步研究旨在确定b1和Alu rna是否在HSV-1感染后抑制Pol II转录中起作用。该提案有三个高度集中的特定目标：1)鉴定Pol ii转录的宿主细胞基因

DESCRIPTION (provided by applicant): 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.