HSV-1 LAT miRNAs: Neurovirulence, reactivation, mechanism

描述(申请人提供)：由于单纯疱疹病毒1型(HSV-1)在三叉神经节(TG)潜伏期重新激活而导致的复发性疱疹可导致单纯疱疹脑炎(HSE)和间质疱疹角膜炎(HSK)。HSE是免疫能力强的个体中最常见的散发性致死性脑炎的原因。同样，单纯疱疹病毒性角膜炎也是导致角膜失明的最常见原因。HSV-1潜伏期相关转录本(LAT)是唯一在潜伏期大量表达的病毒基因，它与病毒的神经毒力及其再激活表型有关，因为该基因组内的各种突变会影响其中的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，这将导致针对疱疹病毒性脑炎和角膜失明的临床治疗的改进。最近，有8个“LAT”microRNAs(MiRNAs)被报道。大多数与已知的影响神经毒力和/或再激活的重要病毒基因重叠。此外，一些miRNAs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。要验证这一假设，需要构建LAT miRNA KO突变体，而由于miRNA序列与关键的HSV-1基因重叠，这一点变得复杂起来。简单地删除miRNA序列将扰乱这些病毒基因，并显著影响与miRNAs无关的病毒功能。通过利用密码子冗余来不改变重叠基因的氨基酸序列，我们改变了其中一个前体miRNAs(H2 MiRNA)中75个核苷酸中的21个。这是在wt(LAT())和LAT(-)基因组背景下完成的。H2 KO突变体不表达H2 miRNA。在初步研究中，两个H2 KO突变体都增加了小鼠的神经毒力，LAT(-)H2 KO突变体也增加了重新激活。Wt-h2KO的重新激活结果尚未公布。这些初步研究有力地表明，H2 miRNA通常参与降低HSV-1的神经毒力和重新激活。我们还发现，LAT miRNAs的表达不需要LAT启动子，这表明一个或多个LAT miRNAs可能解释了LAT启动子缺失突变体仍然支持某些重新激活的能力。如果如预期的那样，我们发现一个或多个LAT miRNAs显著影响神经毒力(或自发再激活表型)，那么对这些miRNAs的操作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突变体并进行体内分析，验证LA miRNAs影响HSV-1神经毒力的假设。2)验证LAT miRNAs影响HSV-1再激活表型的假设。3)验证LAT miRNAs影响神经毒力和/或再激活的机制(S)是通过改变与神经毒力和再激活有关的病毒基因的表达。

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.