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

角膜 HSV-1：LAT 的抗凋亡活性和潜伏期

• 批准号: 7892433
• 负责人: STEVEN L WECHSLER
• 金额: $ 48.98万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7892433
• 关键词: Acute; Amino Acid Sequence; Apoptosis; Apoptotic; Binding; Blindness; Caspase; Cessation of life; Cleaved cell; Clinical; Codon Nucleotides; Complex; Cornea; Development; Disease; Figs - dietary; Functional RNA; Genes; Genetic; Goals; Herpesvirus 1; Herpetic Keratitis; Incidence; Infection; Infectious Agent; Intervention; Knock-out; Knowledge; Lead; Methods; Molecular; Mus; Neurons; Open Reading Frames; Oryctolagus cuniculus; Pathway interactions; Phenotype; Plasmids; Progress Reports; Proteins; RNA; RNA Splicing; Recurrence; Reporting; Severities; Signal Transduction; Small RNA; Structure; Testing; Time; Transcript; Untranslated RNA; Variant; Viral; Viral Genes; Virus; base; cytochrome c; innovation; latency associated transcript; latent infection; mutant; novel; promoter; public health relevance; reactivation from latency

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.

描述（由申请人提供）：病毒重新激活的复发性HSV-1感染是病毒诱导失明的主要原因。我们的总体目标是阐明HSV-1潜伏期反应周期背后的基本分子机制，希望能够发展出一种减少HSV-1重新激活的手段，从而导致HSV-1引起的角膜盲的发生率。 LAT，在潜伏期期间大量转录的唯一病毒基因通过阻断凋亡来增强重新激活表型。由于尚未报告LAT蛋白，因此认为LAT通过非编码RNA起作用。与这个概念相反，我们现在有有力的证据表明在潜伏期期间表达的抗凋亡LAT蛋白（L2）。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延迟相关功能，并且由于L2和RNA-1似乎都具有抗凋亡活性，并且由功能性1St 1.5 Kb的LAT编码，因此我们假设这两者都有助于LAT增强反应活化表型的能力。我们追求这些新颖的创新发现的具体目的包括：1。确认L2（由NTS 487-669编码）和剪接L2的假设是具有抗凋亡活性的真实LAT蛋白，通过：质粒的抗凋亡活性； b）在没有纬度序列的质粒中表达人源化的L2（H-L2）ORF（60 AA）与H-Spliced-L2 ORF（118 AA），并确认其抗凋亡活性； c）将L2和剪接L2的c-末端标记在单独的WT病毒中，并确定其在急性感染中其表达的变化，而潜在感染与重新激活相比。 2。证实L2，剪接L2和RNA-1有助于LAT支持WT重新激活表型的能力，并且至少其中一个在重新激活过程中产生了主要影响，而不是通过以下方式建立延迟： b）构建仅表达H-L2，H-Spliced-L2或RNA-1的突变体，并确认与LAT（ - ）病毒相比，它们具有增加的重新激活表型； c）在建立潜伏期与重新激活过程中阻断L2，剪接L2或RNA-1的表达/功能，并确定对重新激活表型的影响。 3。检验以下假设：L2，剪接L2和RNA-1通过不同的机制干扰凋亡，通过：a）确认它们具有不同的能力来阻断由不同的药物/方法引起的凋亡； b）确认它们阻止外部FAS途径中的不同步骤； c）确定它们是否与不同的凋亡因子结合，表明它们阻止了结合凋亡因子的功能。公共卫生相关性：在美国，单纯疱疹病毒1型（HSV-1）感染是感染剂引起的角膜失明的主要原因，使眼睛HSV-1成为临床上重要的问题。该应用旨在理解HSV-1 LAT基因增强病毒重新激活表型的分子机制。这里获得的知识对于开发有效的临床干预措施的长期目标至关重要，从而降低了复发性眼疱疹性疾病的发生率和/或严重性。