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: 8466973
• 负责人: STEVEN L WECHSLER
• 金额: $ 45.36万
• 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/8466973
• 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; reactivation from latency

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.

病毒再激活引起的HSV-1反复感染是导致病毒致盲的主要原因。 我们的总体目标是阐明HSV-1潜伏期-重新激活背后的潜在分子机制 循环，希望导致开发一种方法来减少HSV-1的重新激活，从而减少 单纯疱疹病毒1型致盲。后来，唯一在潜伏期大量转录的病毒基因增强了 通过阻断细胞凋亡重新激活表型。由于没有LAT蛋白的报道，LAT被认为是 通过非编码的RNA发挥作用。与这个概念相反，我们现在有强有力的证据证明一种抗凋亡药物 在潜伏期内表达的晚期蛋白(L2)。我们也有证据证明第一个小的LAT RNA(RNA-1) 这似乎也有抗细胞凋亡的活性。由于LAT的抗细胞凋亡活性是其最重要的 由于L2和RNA-1似乎都具有抗凋亡活性，并被编码为 通过LAT的功能前1.5 KB，我们假设两者都有助于LAT增强 重新激活表型。我们追求这些新颖创新发现的具体目标包括： 1.确认L2(由NTS 487-669编码)和Spliced-L2是真正的LAT蛋白的假设 通过以下方式具有抗凋亡活性：a)人源化L2 ORF NT序列而不改变L2氨基酸 表达LAT功能前1.5kb的质粒中的序列及其保留 抗细胞凋亡活性；b)人源化L2(h-L2)ORF(60aa)与h-剪接L2ORF(118aa)的表达 在没有LAT侧翼序列的质粒中，并确认它们的抗凋亡活性；c)标记C- 分离的wt病毒中L2和与myc拼接的L2末端及其变化的测定 在急性感染、潜伏感染和重新激活期间的表达。 2.确认L2、剪接L2和RNA-1有助于LAT支持wt的能力的假设 重新激活表型，并且至少其中之一在重新激活过程中发挥主要影响，而不是 通过以下方式建立潜伏期：a)构建敲除突变体并确认其重新激活程度降低 表型；b)构建仅表达h-L2、h-拼接-L2或RNA-1的突变体，并证实它们 与LAT(-)病毒相比具有更多的重新激活表型；c)阻断L2的表达/功能， 剪接的L2，或RNA-1，在潜伏期建立与重新激活，并确定对 重新激活表型。 3.检验L2、剪接L2和RNA-1通过不同机制干预细胞凋亡的假说 通过：a)证实它们具有不同的阻断不同试剂/方法诱导的细胞凋亡的能力； B)确认它们阻断了外在Fas途径中的不同步骤；c)确定它们是否与不同的 凋亡因子，提示它们阻断了结合的凋亡因子的功能。

项目成果

A speculated ribozyme site in the herpes simplex virus type 1 latency-associated transcript gene is not essential for a wild-type reactivation phenotype.

1 型单纯疱疹病毒潜伏相关转录基因中推测的核酶位点对于野生型再激活表型并不是必需的。

• DOI: 10.1080/13550280802275912
• 发表时间: 2008
• 期刊: Journal of neurovirology
• 影响因子: 3.2
• 作者: Carpenter,Dale;Singh,Sukhpreet;Osorio,Nelson;Hsiang,Chinhui;Jiang,Xianzhi;Jin,Ling;Jones,Clinton;Wechsler,StevenL
• 通讯作者: Wechsler,StevenL

Activators of potassium M currents have anticonvulsant actions in two rat models of encephalitis.

钾 M 电流激活剂在两种脑炎大鼠模型中具有抗惊厥作用。

• DOI: 10.1016/j.ejphar.2006.10.025
• 发表时间: 2007
• 期刊: European journal of pharmacology
• 影响因子: 5
• 作者: Solbrig,MarylouV;Adrian,Russell;Wechsler,StevenL;Koob,GeorgeF
• 通讯作者: Koob,GeorgeF

Herpes simplex virus type 1 latency-associated transcript inhibits apoptosis and promotes neurite sprouting in neuroblastoma cells following serum starvation by maintaining protein kinase B (AKT) levels.

• DOI: 10.1099/vir.0.015719-0
• 发表时间: 2010-04
• 期刊: The Journal of general virology
• 作者: Sumin Li;D. Carpenter;C. Hsiang;S. Wechsler;Clinton Jones

Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice.

• DOI: 10.1080/13550280802216510
• 发表时间: 2008-10
• 期刊: Journal of neurovirology
• 影响因子: 3.2
• 作者: Jin L;Carpenter D;Moerdyk-Schauwecker M;Vanarsdall AL;Osorio N;Hsiang C;Jones C;Wechsler SL
• 通讯作者: Wechsler SL