HSV-1 LAT miRNAs: Neurovirulence, reactivation, mechanism

描述（由申请人提供）：复发性疱疹，由于三叉神经节（TG）潜伏期的单纯疱疹病毒1型（HSV-1）重新激活，可引起单纯疱疹病毒性脑炎（HSE）和疱疹性角膜基质炎（HSK）。HSE是免疫能力正常的个体中散发性致死性脑炎的最常见原因。类似地，HSK是由于感染剂导致的角膜失明的最常见原因。HSV-1潜伏期相关转录物（LAT）是潜伏期期间大量表达的唯一病毒基因，其参与病毒的神经毒力和其再活化表型，因为该基因组基因座内的各种突变体影响一个或另一个或两者。我们的长期目标是了解HSV-1神经毒力，潜伏期和再激活背后的分子机制，这将导致改善针对疱疹性脑炎和角膜失明的临床治疗。最近报道了8种“LAT”microRNAs（miRNAs）。大多数重叠的重要病毒基因已知影响神经毒力和/或再激活。此外，一些miRNA影响这些基因的表达。我们假设LAT miRNAs影响神经毒力和/或再激活表型。验证这一假设需要构建LAT miRNA KO突变体，这由于miRNA序列与关键HSV-1基因重叠的事实而变得复杂。miRNA序列的简单缺失将破坏这些病毒基因，并显著影响与miRNA无关的病毒功能。通过利用密码子冗余不改变重叠基因的氨基酸序列，我们改变了一个前体miRNA（H2 miRNA）中75个nt中的21个。这是在wt（LAT（+））和LAT（-）基因组背景上进行的。H2 KO突变体不表达H2 miRNA。在初步研究中，两种H2 KO突变体在小鼠中的神经毒力增加，LAT（-）H2 KO突变体也增加了再活化。尚未获得wt-H2 KO的再活化结果。这些初步研究强烈表明，H2 miRNA通常参与降低HSV-1神经毒力和再活化。我们还发现，LAT miRNA不需要LAT启动子来表达，这表明一个或多个LAT miRNA可能解释了LAT启动子缺失突变体仍然支持一些再激活的能力。如果如预期的那样，我们发现一种或多种LAT miRNAs显著影响神经毒力（或自发再激活表型），那么操纵这些miRNAs将是未来临床干预的有用靶点。我们的具体目标包括：1）通过构建LAT miRNA KO突变体并在体内分析它们来检验LA miRNA影响HSV-1神经毒力的假设。2)检验LAT miRNA影响HSV-1再活化表型的假设。3)验证LAT miRNA影响神经毒力和/或再活化的机制是改变参与神经毒力和再活化的病毒基因的表达的假设。

DESCRIPTION (provided by applicant): Recurrent herpes, due to reactivation of herpes simplex virus type 1 (HSV-1) from latency in the trigeminal ganglia (TG) can cause herpes simplex encephalitis (HSE) and herpes stromal keratitis (HSK). HSE is the most common cause of sporadic lethal encephalitis in immune competent individuals. Similarly, HSK is the most common cause of corneal blindness due to an infectious agent. The HSV-1 latency associated transcript (LAT), the only viral gene abundantly expressed during latency, is involved in both the virus' neurovirulence and its reactivation phenotype, since various mutants within this genomic locus affect one or the other or both. Our long term goal is to understand the molecular mechanisms behind HSV-1 neurovirulence, latency, and reactivation, which should lead to improved clinical therapies directed against herpes encephalitis and corneal blindness. Recently, 8 "LAT" microRNAs (miRNAs) were reported. Most overlap important viral genes known to affect neurovirulence and/or reactivation. In addition, some of the miRNAs affect expression of these genes. We hypothesize that LAT miRNAs influence neurovirulence and/or the reactivation phenotype. Testing this hypothesis requires construction of LAT miRNA KO mutants, which is complicated by the fact that the miRNA sequences overlap critical HSV-1 genes. Simple deletion of the miRNA sequences would disrupt these viral genes and significantly impact viral functions unrelated to the miRNAs. By making use of codon redundancy to not alter the amino acid sequence of an overlapping gene, we changed 21 of the 75 nts in one of the precursor miRNAs (the H2 miRNA). This was done on both a wt (LAT(+)) and a LAT(-) genomic background. The H2 KO mutants expressed no H2 miRNA. In preliminary studies both H2 KO mutants had increased neurovirulence in mice, and the LAT(-) H2 KO mutant also had increased reactivation. Reactivation results for the wt-H2 KO are not yet available. These preliminary studies strongly suggest that the H2 miRNA is normally involved in decreasing HSV-1 neurovirulence and reactivation. We also found that the LAT miRNAs do not require the LAT promoter for their expression, suggesting that one or more LAT miRNAs may account for LAT promoter deletion mutants' ability to still support some reactivation. If, as expected, we find that one or more of the LAT miRNAs significantly affects neurovirulence (or the spontaneous reactivation phenotype), manipulation of those miRNAs would be a useful target for future clinical interventions. Our Specific Aims include: 1) Test the hypothesis that LA miRNAs influence HSV-1 neurovirulence by constructing LAT miRNA KO mutants and analyzing them in vivo. 2) Test the hypothesis that the LAT miRNAs influence the HSV-1 reactivation phenotype. 3) Test the hypothesis that the mechanism(s) by which LAT miRNAs influence neurovirulence and/or reactivation is to alter expression of viral genes involved in neurovirulence and reactivation.