Modulation and Utilization of RNA Polymerase III by Herpes Simplex Virus

单纯疱疹病毒基因组除了编码大量非编码RNA外，还编码约90个蛋白质编码基因的mRNAs。这些基因的组合由RNA polII以顺序的方式转录。它们的表达时间取决于它们的启动子中细胞转录因子的顺式作用位点，它们对病毒转录因子的要求，以及病毒DNA复制的状态。多种病毒蛋白修饰或隔离PolII转录机制的元件，以实现病毒基因组的强劲转录，从而减弱细胞mRNA的转录。我们已经发现RNA polIII及其一些转录因子也与HSV基因组结合。RNA polIII转录tRNAs和其他细胞非编码RNA。我们还表明，在对病毒激活剂的要求和病毒DNA复制的影响方面，PolIII与病毒基因组的结合方面类似于PolII。然而，与PolII不同的是，我们发现感染会增加特定细胞PolIII转录单位的转录，即tRNA基因。我们假设RNA PolIII在HSV基因组上发挥着转录非编码RNA的功能，并且PolIII在基因组上的功能受到一些相同的顺式和反式作用元件的调节，这些元件调节基因组的PolII转录。我们进一步假设，HSV感染通过改变与细胞POLIII启动子结合的POLIII转录因子的丰度或活性来诱导特定类型的POLIII转录。针对这些假设，本文提出了两个具体的目标。我们将确定PolIII与HSV基因组和由此产生的PolIII转录本相关联的基础，并确定HSV如何影响细胞PolIII转录。其他DNA病毒编码PolIII转录本；然而，这是第一个表明PolIII与HSV基因组相关的研究。可能已知的HSV非编码RNA或尚未发现的非编码RNA是由PolIII转录的。其他病毒也已知能刺激PolIII的转录。与腺病毒或EBV一样，HSV对PolIII转录的激活可能促进病毒基因组的PolIII转录，和/或为病毒蛋白编码mRNAs的翻译提供最佳水平的tRNAs。这项探索性拨款将确定病毒和感染细胞的RNA PolIII转录组，以及病毒感染调节PolIII转录的机制，作为更深入研究PolIII与HSV相互作用及其对病毒感染的意义的前奏。

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.