Suppression of RNA Interference by Herpes Simplex Virus

描述(申请人提供)：真核细胞已经发展出天生的机制来保护它们免受病毒和外来核酸的入侵，其中大多数涉及沉默基因表达。其中一种适应性先天反应是RNA干扰(RNAi)。RNAi途径在几乎所有真核生物中都高度保守，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引导的DNA甲基化、组蛋白H3K9甲基化和异染色质的建立。在植物中，RNAi是一种重要的适应性防御，由病毒诱导并以病毒为靶标。植物病毒通过产生抑制该途径组成部分的蛋白质来对抗这种攻击，而这些沉默抑制物最初被确定为致病决定因素。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的方法的敏感性支持这一观点。病毒沉默抑制子是发展抗病毒和疫苗治疗的有效靶点，但这种抑制子在哺乳动物病毒中的存在尚不清楚。单纯疱疹病毒(HSV)在细胞中有效地建立其转录程序，在其中进行高效复制，但病毒基因的转录在潜伏期基本上是沉默的。HSV在允许细胞中避免沉默的有效性使HSV成为编码沉默抑制子的有力候选者。人们假设HSV编码一种或多种蛋白质，这些蛋白质抑制RNAi途径，以便进行高效复制。野生型和突变型HSV诱导RNAi的能力将取决于病毒特异性小干扰RNA(SiRNA)的出现，该方法采用候选筛选方法和全局方法从感染细胞中克隆小dsRNA。在目标2中，将使用瞬时沉默系统来识别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.