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

描述(申请人提供)：单纯疱疹病毒1型(HSV-1)在感染哺乳动物细胞后的几个小时内会引起宿主细胞转录程序的变化。HSV-1感染后，RNA聚合酶II(POL II)对蛋白质编码基因的转录有很大程度的抑制，但这种抑制的机制尚不完全清楚。有人假设，在病毒感染过程中上调的特定非编码RNA(NcRNAs)在HSV-1感染后作为Pol II转录的全局、转录抑制因子。被认为可以作为抑制物的特定ncRNAs是小鼠B2 RNA和人类Alu RNA，它们分别是由RNA聚合酶III从散布在小鼠和人类基因组中的短散布元件(Sine)转录而来的。这一假说是基于先前对热休克期间发生的mRNA转录的全局抑制的研究；B2和Alu RNAs分别被发现在小鼠和人类细胞热休克后直接抑制Pol II的转录。本文描述的初步研究试图确定B2和Alu RNAs是否在HSV-1感染后抑制POL II转录中发挥作用。该提案有三个高度集中的具体目标。1)鉴定POL II转录的宿主细胞基因 通过测量病毒感染前后基因组中Pol II占有率的变化，抑制了HSV-1感染小鼠和人类细胞后的表达。2)通过敲除ncRNAs，并测定感染前后对Pol II在基因组中占位的影响，确定B2RNA和Alu RNA是否参与了HSV-1感染后宿主细胞Pol II转录的抑制。3)利用两种已建立的基于细胞的分析方法，确定B2和Alu RNA是否与POL II和HSV-1感染反应中被抑制基因的启动子相关联。这项拟议的研究将确定B2和Alu RNA是否介导HSV-1诱导的宿主细胞转录抑制。在检验这一假设时，将在全基因组范围内了解HSV-1感染如何重新编程Pol II在mRNA基因上的占据，以及HSV-1如何影响B2RNA和Alu RNA的活性。这些先导性研究的结果将使人们能够对HSV-1感染、连续的ncRNA转录和功能、宿主细胞转录和病毒基因转录之间的相互作用进行新的研究，这将是未来R01拨款申请的重点。最后，由于已知B2RNA和Alu RNA水平在被其他病毒感染后和对其他生物过程的反应中增加，我们的研究可以为这些ncRNAs在多种情况下如何控制转录提供一般的见解。

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.