Suppression of RNA Interference by Herpes Simplex Virus

描述（由申请人提供）：真核细胞已经开发出保护其免受病毒和外源核酸侵袭的先天机制，其中大多数涉及基因表达的沉默。一种这样的适应性先天反应是RNA干扰（RNAi）。RNAi途径在几乎所有的真核生物中都是高度保守的，并且参与了多种对基因表达产生负面影响的现象，包括RNA沉默、microRNA加工、RNA指导的DNA甲基化、组蛋白H3K9甲基化和异染色质的建立。在植物中，RNAi是由病毒诱导并靶向病毒的一种重要的适应性防御。植物病毒通过产生抑制该途径组分的蛋白质来对抗这种攻击，这些沉默抑制子首先被鉴定为致病性决定因子。哺乳动物病毒很可能也必须抑制RNAi，最近证明它们对基于RNAi的方法的易感性支持了这一观点。病毒沉默抑制子代表了开发抗病毒和疫苗治疗剂的有效靶点，但在哺乳动物病毒中存在此类抑制子尚不清楚。单纯疱疹病毒（HSV）在细胞中建立其转录程序是有效的，在细胞中它可以有效地复制，但病毒基因转录在潜伏期内基本上是沉默的。HSV避免在允许细胞中沉默的有效性使得HSV成为编码沉默抑制子的强候选者。据推测，HSV编码一种或多种抑制RNAi途径的蛋白质，以便有效复制。野生型和突变型HSV诱导RNAi的能力将通过使用候选筛选方法和从感染细胞克隆小dsRNA的全局方法的病毒特异性小干扰RNA（siRNA）的出现来确定。在目标2中，瞬时沉默系统将用于鉴定由HSV编码的沉默抑制子。

DESCRIPTION (provided by applicant): Eukaryotic cells have developed innate mechanisms to protect them from invasion by viruses and foreign nucleic acids, most of which involve silencing of gene expression. One such adaptive innate response is RNA interference (RNAi). The RNAi pathway is highly conserved in almost all eukaryotes, and is involved in a variety of phenomena that negatively impact gene expression, including RNA silencing, microRNA processing, RNA-directed DNA methylation, histone H3K9 methylation, and the establishment of heterochromatin. In plants, RNAi is an important adaptive defense which is induced by and targets viruses. Plant viruses counter this attack by producing proteins that suppress components of the pathway, and these silencing suppressors were first identified as pathogenicity determinants. It is likely that mammalian viruses must also suppress RNAi, and their recently demonstrated susceptibility to RNAi-based approaches supports this view. Viral silencing suppressors represent potent targets for the development of antiviral and vaccine therapeutics, but the presence of such suppressors among mammalian viruses is unknown. Herpes simplex virus (HSV) is effective in establishing its transcription program in cells in which it replicates productively, but viral gene transcription is largely silenced during latency. The effectiveness with which HSV avoids being silenced in permissive cells makes HSV a strong candidate for encoding a silencing suppressor. It is hypothesized that HSV encodes one or more proteins that suppress the RNAi pathway in order to productively replicate. The ability of wild-type and mutant HSV to induce RNAi will be determined by the appearance of virus-specific small interfering RNA (siRNA) using a candidate screening approach and a global approach to clone small dsRNAs from infected cells. In aim 2, a transient silencing system will be used to identify silencing suppressors encoded by HSV.