Corneal HSV-1: LAT's Anti-Apoptosis Activity and Latency

描述（由申请人提供）：由于病毒再激活导致的复发性HSV-1感染是病毒性失明的主要原因。我们的总体目标是阐明HSV-1潜伏-再激活周期背后的潜在分子机制，希望能找到减少HSV-1再激活的方法，从而减少HSV-1诱导角膜失明的发生率。LAT是唯一在潜伏期大量转录的病毒基因，通过阻断细胞凋亡来增强再激活表型。由于没有LAT蛋白的报道，LAT被认为是通过非编码RNA起作用的。与这一观点相反，我们现在有强有力的证据表明抗凋亡LAT蛋白（L2）在潜伏期表达。我们也有证据表明，第一个小的LAT RNA （RNA-1）似乎也具有抗凋亡活性。由于LAT的抗凋亡活性是其最重要的潜伏期相关功能，并且L2和RNA-1似乎都具有抗凋亡活性，并且由LAT的第一个1.5 Kb功能性编码，我们假设它们都有助于LAT增强再激活表型的能力。我们追求这些新颖创新发现的具体目标包括：1。通过以下方法证实L2（由nts 487-669编码）和剪接L2是具有抗凋亡活性的真实LAT蛋白的假设：a)在不改变表达LAT第1个1.5 Kb功能的质粒中L2氨基酸序列的情况下，人源化L2 ORF nt序列，并确认质粒的抗凋亡活性保留；b)在无LAT侧链的质粒中分别表达人源化的L2 (h-L2) ORF （60 aa）和h-剪接的L2 ORF (118 aa)，并证实其抗凋亡活性；c)在不同的wt病毒中用myc标记L2的c末端和剪接L2，并确定它们在急性感染、潜伏感染和再激活期间的表达变化。2. 证实L2、剪接L2和RNA-1有助于LAT支持wt再激活表型的假设，并且至少其中一个在再激活过程中发挥其主要影响，而不是建立潜伏期：a)构建敲除突变体并确认其减少的再激活表型；b)构建仅表达h-L2、h-剪接- l2或RNA-1的突变体，并证实与LAT（-）病毒相比，它们具有增加的再激活表型；c)在潜伏期和再激活建立过程中阻断L2、剪接L2或RNA-1的表达/功能，并确定对再激活表型的影响。3. 验证L2、剪接L2和RNA-1通过不同机制干扰细胞凋亡的假设：a)证实它们对不同药物/方法诱导的细胞凋亡具有不同的阻断能力；b)证实它们阻断了外源性Fas通路的不同步骤；c)确定它们是否与不同的凋亡因子结合，表明它们阻断了结合的凋亡因子的功能。公共卫生相关性：在美国，复发性眼部单纯疱疹病毒1型（HSV-1）感染是由感染性病原体引起的角膜失明的主要原因，使眼部HSV-1成为一个重要的临床问题。该应用旨在了解HSV-1 LAT基因增强病毒再激活表型的分子机制。这里获得的知识对于制定针对LAT的有效临床干预措施的长期目标至关重要，从而降低复发性眼疱疹病的发病率和/或严重程度。

DESCRIPTION (provided by applicant): Recurrent HSV-1 infection as a result of viral reactivation is a major cause of viral induced blindness. Our overall goal is elucidation of the underlying molecular mechanisms behind the HSV-1 latency-reactivation cycle, hopefully leading to development of a means for reducing HSV-1 reactivation and hence the incidence of HSV-1 induced corneal blindness. LAT, the only viral gene abundantly transcribed during latency enhances the reactivation phenotype by blocking apoptosis. Since no LAT protein has been reported, LAT is thought to function via a noncoding RNA. In contrast to this notion, we now have strong evidence for an anti-apoptotic LAT protein (L2) that is expressed during latency. We also have evidence for the first small LAT RNA (RNA-1) that also appears to have anti-apoptosis activity. Since LAT's anti-apoptosis activity is its most important latency related function and since L2 and RNA-1 both appear to have anti-apoptosis activity and are encoded by the functional 1st 1.5 Kb of LAT, we hypothesize that both contribute to LAT's ability to enhance the reactivation phenotype. Our Specific Aims to pursue these novel innovative findings include: 1. Confirm the hypotheses that L2 (encoded by nts 487-669) and spliced-L2 are authentic LAT proteins with anti-apoptosis activity by: a) Humanizing the L2 ORF nt sequence without changing the L2 amino acid sequence in a plasmid expressing the functional 1st 1.5 Kb of LAT and confirming retention of the plasmid's anti-apoptosis activity; b) Expressing the humanized L2 (h-L2) ORF (60 aa) vs. the h-spliced-L2 ORF (118 aa) in plasmids with no LAT flanking sequences and confirming their anti-apoptosis activity; c) Tagging the C- terminus of L2 and spliced-L2 with myc in separate otherwise wt viruses and determining changes in their expression during acute infection vs. latent infection vs. reactivation. 2. Confirm the hypothesis that L2, spliced-L2, and RNA-1 contribute to LAT's ability to support the wt reactivation phenotype and that at least one of them exerts its main influence during reactivation rather than establishment of latency by: a) Constructing knock out mutants and confirming their reduced reactivation phenotype; b) Constructing mutants that express just h-L2, h-spliced-L2, or RNA-1 and confirming that they have an increased reactivation phenotype compared to LAT(-) viruses; c) Blocking expression/function of L2, spliced-L2, or RNA-1 during establishment of latency vs. reactivation, and determining the effect on the reactivation phenotype. 3. Test the hypothesis that L2, spliced-L2, and RNA-1 interfere with apoptosis via different mechanisms by: a) Confirming that they have differing abilities to block apoptosis induced by different agents/methods; b) Confirming that they block different steps in the extrinsic Fas pathway; c) Determining if they bind to different apoptotic factors, suggesting that they block function of the bound apoptotic factor. PUBLIC HEALTH RELEVANCE: In the US recurrent ocular herpes simplex virus type 1 (HSV-1) infection is the leading cause of corneal blindness due to an infectious agent, making ocular HSV-1 a clinically important problem. This application is directed at understanding the molecular mechanisms by which the HSV-1 LAT gene enhances the virus' reactivation phenotype. The knowledge gained here will be critical for the longer term goal of developing efficacious clinical interventions that target LAT and thereby decrease the incidence and/or severity of recurrent ocular herpetic disease.