HSV Drug Targets, Gene Expression, and Latency

HSV 药物靶点、基因表达和潜伏期

• 批准号: 7676710
• 负责人: David Coen
• 金额: $ 45.39万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7676710
• 关键词: Acyclovir; Affect; Alleles; Antigen Presentation; Antiviral Agents; Authorization documentation; Bacterial Artificial Chromosomes; Behavior; Biological; Biological Assay; Biology; Boston; Cell Line; Cell Nucleus; Chinese People; Chromatin; Class; Clinical; Code; Collaborations; Cultured Cells; DNA biosynthesis; DNA chemical synthesis; Detection; Disclosure; Disease; Drug Delivery Systems; Drug resistance; Enzymes; Frameshift Mutation; Ganglia; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Grant; Herpesviridae; Human Resources; ICP47; Immune; Implantable Pump; Infection; Instruction; Interferons; Interphase Cell; Knockout Mice; Last Name; Lead; Light; Maintenance; Maps; Measures; Mediating; Methods; MicroRNAs; Mus; Mutation; Myxoid cyst; Names; Nervous system structure; Neurons; Numbers; Pathogenicity; Pharmacotherapy; Postdoctoral Fellow; Principal Investigator; Property; Protein Biosynthesis; Proteins; Registries; Regulation; Relative (related person); Repression; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Science; Simplexvirus; Technology; Testing; Thymidine Kinase; Time; Transcript; VP 16; Vaccines; Viral; Viral Gene Expression Regulation; Viral Genes; Viral Proteins; Virion; Virus; Virus Diseases; Virus Latency; base; cell type; fascinate; human embryonic stem cell; lytic gene expression; medical schools; member; mutant; novel; prevent; programs; reactivation from latency; research study; viral DNA

The long-term objectives of this project have been to investigate the roles of viral DMA replication, replication proteins that serve as targets for antiviral drugs such as acyclovir, and gene expression in the interaction of herpes simplex virus (HSV) with the mammalian nervous system, especially virus latency. Latency is the most fascinating biological property of the virus and its most important clinical feature as it permits the virus to remain in the host and subsequently reactivate to cause disease. Understanding HSV latency is sure to reveal novel features of HSV and the nervous system. Such information is highly relevant to antiviral drugs that target DMA replication, vaccines, and potential agents to cure HSV infections. The proposed research will study the mechanisms by which acyclovir-resistant mutants retain pathogenicity, in particular the ability to reactivate from latency (aim 1). The relative contributions of low thymidine kinase (TK) expression and reversion will be assessed in experiments in which acyclovir will be administered to mice using implantable pumps during establishment of latency. Bacterial artificial chromosome methods will be used to map alleles of clinical isolates that compensate for loss of TK in reactivation. How viral gene expression is regulated during latency will be explored (aim 2). Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assays will be used to measure expression of different classes of transcripts during latency and reactivation. Knock-out mice will be used to test the hypothesis that immune effectors such as inteferon y repress the expression of viral genes. Why late transcripts accumulate during reactivation in the presence of acyclovir will be explored. A third set of studies will investigate whether the latency-associated transcript (LAT) locus represses viral gene expression through short intefering RNAs and microRNAs that can be expressed from this locus. The presence of these RNAs will be assayed in latently infected ganglia and, in collaboration with project 1, chromatin. Mutant viruses will be constructed to test the importance of these mechanisms for repression. Changes in host gene expression, which may affect latency, will also be investigated (aim 3). Array technologies, RT-PCR, and knockout mice will be used to identify whether observed changes in host gene expression are due to immune effectors such as interferon y. Viral mutants will be used to examine the role of viral factors (e.g. microRNAs) in these changes. The proposed experiments should shed light on viral and cellular factors involved in the establishment, maintenance, and reactivation of HSV latency. Latency is why herpesviruses cause diseases for which there are no cures. Understanding how latency is established, maintained, and reactivated could lead to a cure for HSV. Harvard Medical School, Boston, MA PHS 398(Rev. 09/04) Page Form Page 2 000135 Project 2 Principal Investigator/Program Director (Last, First, Middle): Schaffei", Priscilla A. KEY PERSONNEL. See instructions. Use continuation pages as neededto provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Coen, Donald M. Harvard MedicalSchool PrincipalInvestigator Griffiths, Anthony Harvard Medical School Research Fellow Kramer, Martha F. Harvard MedicalSchool Research Associate Pesola, Jean Harvard MedicalSchool Research Fellow OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Moazed, Danesh Harvard Medical School Consultant Wang, Xiujie Chinese Academyof Sciences jCollaboratoij Human Embryonic Stem Cells E3 No Q Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://sterncells.nlh.gov/reaistrv/index.asp. Usecontinuation pages as needed. If a specific line cannot be referencedat this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. Yes No PHS 398 (Rev. 09/04) Page Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. 000136 Project 2 Principal Investigator/ProgramDirector (Last, First, Middle): Schaffer, PriSCJIla Ann A. Specific Aims The long-term objectives of this project have been to investigate the roles of viral DNA replication and replication proteins (especially those that serve as targets for antiviral drugs), and gene expression in the interaction of herpes simplex virus (HSV) with the mammalian nervous system. These studies have implications for the biology of the virus and the nervous system, and for antiviral drugs and drug resistance. The goals are a subset of the overall goal of the program, which is to identify viral and cellular factors involved in the establishment/maintenance, and reactivation of HSV latency. The studies are very much in collaboration with the other members of the program. During the previous grant period, studies of thymidine kinase (tk) mutations that cause acyclovir-resistance (ACVr) in drug-resistant clinical isolates, have illuminated three different general ways by which the virus may evade drug therapy, yet retain pathogenicity. Studies detecting and quantifying HSV transcripts have raised the possibility of a specific block in late gene expression during latency, and revealed unexpected behaviors of late gene transcripts during reactivation. Other results from these studies and our recent detection of HSV- encoded microRNAs (miRNAs), have suggested mechanisms for how the latency-associated transcript (LAT) locus mediates repression during latency. Studies using array technology have revealed specific changes in neuronal gene expression in latently infected ganglia. These previous studies provide the basis for the current proposal whose specific aims are: 1. To study how ACV tk mutants from clinical isolates retain pathogenicity, focusing on the interplay of low level TK activity with reversion of frameshift mutations, and on alleles in genes other than tk that permit reactivation from latency in the absence of TK. 2. To investigate regulation of viral gene expression during HSV latency in mice, in particular to investigate regulation of late gene expression during latency and reactivation, and how repression may be mediated by the LAT locus and by immune effectors such as interferon (IFN) y. 3. To study changes in host gene expression during HSV latency in mice and how host and viral factors effect these changes. B. Background and Significance. The interaction of HSV with the mammalian nervous system that results in virus latency is the most fascinating biological property of the virus and its most important clinical feature. How does a virus that is so accomplished at productive infections completely alter its program of gene expression and replication to establish a silent, yet reactivatable infection of a neuron? Answering this question seems certain to uncover novel aspects of HSV and the nervous system. Such information is highly relevant to strategies to prevent and treat HSV infections and might even lead to agents to cure HSV infections. Moreover, studies of virus mutants with alterations in TK not only shed light on latency, but on mechanisms of gene expression and, because TK is a drug target, have implications for the use of antiviral drugs. Background information relevant to this proposal will now be reviewed: 1. HSV productive cycle ("lytic") gene expression. HSV is a large, enveloped virus containing a -150 kbp linear genome whose sequence predicts -70 protein coding genes (84). After entry of HSV DNA into the nucleus of cultured cells, a cascade of gene expression ensues [unless otherwise cited, see (97)]. Transcription of the first class of genes to be expressed (immediate early, IE, a) is stimulated by a virion component, VP16, and does not require prior viral protein synthesis. IE gene products include ICP 4, 27, 0, and 22, which are regulators of gene expression, and ICP47, which antagonizes antigen presentation. ICP4 and 27 are essential for viral replication. ICPO, ICP22, and ICP47 are non-essential. However, ICPO is especially important at low multiplicities of infection in resting cells and ICP22 is more important in some cell types than in others. The subsequent two classes of genes to be expressed are termed early (E, (3) and late (L, Y),respectively, based on when in the infectious cycle their expression peaks. E genes mainly encode proteins involved in DNA replication. These include enzymes that directly participate in DNA synthesis and are PHS 398/2590 (Rev. 09/04) Page Continuation Format Page 000137

该项目的长期目标是研究病毒DMA复制的作用， 复制蛋白作为抗病毒药物（如阿昔康维尔）的靶标，以及在基因表达中的基因表达 单纯疱疹病毒（HSV）与哺乳动物神经系统，尤其是病毒潜伏期的相互作用。 潜伏期是该病毒最迷人的生物学特性及其最重要的临床特征 允许病毒留在宿主中，然后重新激活以引起疾病。了解HSV 延迟肯定会揭示HSV和神经系统的新颖特征。这些信息高度相关 靶向DMA复制，疫苗和潜在药物以治愈HSV感染的抗病毒药物。 拟议的研究将研究抗性抗性突变体的机制 致病性，特别是从潜伏期中重新激活的能力（AIM 1）。低的相对贡献 胸苷激酶（TK）的表达和回归将在Acyclovir的实验中进行评估 在建立潜伏期期间，使用植入泵对小鼠进行管理。细菌人造 染色体方法将用于绘制临床分离株的等位基因，以补偿TK的损失 重新激活。将如何探索在潜伏期期间调节病毒基因表达（AIM 2）。定量 逆转录酶 - 聚合酶链反应（RT-PCR）测定将用于测量 延迟和重新激活期间的不同类别的转录本。淘汰小鼠将用于测试 假设免疫效应子（例如inteferon y）抑制病毒基因的表达。为什么迟到 将探索在acyclovir存在下重新激活期间积累的转录本。第三组研究 将研究潜伏相关的转录（LAT）基因座是否抑制病毒基因表达 通过简短的整数RNA和microRNA，可以从该基因座表达。这些的存在 RNA将在潜在感染的神经节中进行测定，并与Project 1 Chromatin合作。突变体 将构建病毒以检验这些机制在抑制的重要性。主机的变化 可能影响潜伏期的基因表达也将进行研究（AIM 3）。阵列技术，RT-PCR， 和敲除小鼠将用于确定观察到的宿主基因表达的变化是否是由于 免疫效应子，例如干扰素y。病毒突变体将用于检查病毒因子的作用（例如 microRNA）在这些变化中。提出的实验应阐明病毒和细胞因子 参与HSV潜伏期的建立，维护和重新激活。 延迟是为什么疱疹病毒引起无法治愈的疾病的原因。了解潜伏期 建立，维护和重新激活可能会导致治疗HSV。 马萨诸塞州波士顿哈佛医学院 PHS 398（修订版09/04）页面2页 000135 项目2 首席研究员/计划主管（最后，第一，中间）：Schaffei”，Priscilla A. 关键人员。请参阅说明。根据需要使用延续页面，以下面显示的格式提供所需的信息。 从首席研究员开始。按字母顺序列出所有其他关键人员，首先姓氏。 名称ERA CONSONS用户名组织在项目中的角色 Coen，Donald M. Harvard Medicalschool PrincivealIvestigator 格里菲斯，安东尼·哈佛医学院研究员 克莱默（Kramer），玛莎（Martha F.）哈佛医学院研究助理 佩索拉，让·哈佛医学院研究员 其他重要的贡献者 姓名组织角色 Moazed，Danesh Harvard医学院顾问 王，xiujie中国科学学院jcollaboratoij 人类胚胎干细胞E3否Q是 如果所提出的项目涉及人类胚胎干细胞，请从以下列表中列出特定细胞系的注册数： http://sterncells.nlh.gov/reaistrv/index.asp。根据需要进行用户关注页。 如果这次无法引用特定行，请包括一个将使用注册表中的陈述。 细胞系 披露许可声明。仅适用于SBIR/STTR。请参阅SBIR/STTR说明。是否 PHS 398（Rev. 09/04）页面2页面第2页 在整个过程中连续编号以下页面 应用程序。请勿使用4A，4B等后缀。 000136 项目2首席调查员/程序导演（最后，第一，中间）：Schaffer，Priscjila Ann A.具体目标 该项目的长期目标是研究病毒DNA复制的作用和 复制蛋白（尤其是作为抗病毒药物靶标的蛋白）和基因表达 疱疹病毒（HSV）与哺乳动物神经系统的相互作用。这些研究有 对病毒和神经系统的生物学以及抗病毒药物和耐药性的影响。 目标是该计划的整体目标的子集，即确定涉及的病毒和细胞因素 在建立/维护和HSV潜伏期的重新激活中。研究非常重要 与该计划的其他成员合作。 在上一个赠款期间，胸苷激酶（TK）突变的研究引起了抗性抗性抗性 （ACVR）在耐药的临床分离株中，已经照亮了病毒可以通过的三种一般方式 逃避药物治疗，但保留致病性。检测和量化HSV转录本的研究已提高 潜伏期期间基因表达中特定块的可能性，并揭示了意想不到的行为 重新激活过程中的晚期基因转录本。这些研究的其他结果以及我们最近发现HSV- 编码的microRNA（miRNA）提出了有关潜伏相关转录（LAT）的机制 基因座介导延迟期间的抑制作用。使用阵列技术的研究揭示了特定的变化 潜在感染神经节中神经元基因表达。这些先前的研究为当前提供了基础 提案的具体目标是： 1。研究临床分离株的ACV TK突变体如何保留致病性，以关注低的相互作用 TK活性，带有移码突变的重新转换，以及允许TK以外的其他基因等位基因 在没有TK的情况下潜伏的重新激活。 2。研究小鼠HSV潜伏期期间病毒基因表达的调节，特别是研究 调节延迟和重新激活期间晚期基因表达，以及如何介导的抑制作用 LAT基因座和免疫效应子，例如干扰素（IFN）y。 3。研究小鼠HSV潜伏期期间宿主基因表达的变化以及宿主和病毒因素如何效应 这些变化。 B.背景和意义。 HSV与导致病毒潜伏期的哺乳动物神经系统的相互作用最多 该病毒的引人入胜的生物学特性及其最重要的临床特征。这样的病毒如何 在生产感染下完成的基因表达和复制程序完全改变了 建立神经元的沉默但可重复的感染？回答这个问题似乎肯定会发现 HSV和神经系统的新颖方面。这些信息与预防策略高度相关和 治疗HSV感染，甚至可能导致药物治愈HSV感染。此外，对病毒突变体的研究 随着TK的改变，不仅阐明了潜伏期，而且还阐明了基因表达的机制，并且因为TK 是药物靶标，对使用抗病毒药有影响。背景信息与此相关 现在将审查提案： 1。HSV生产循环（“裂解”）基因表达。 HSV是一种包含-150 kbp的大型包围病毒 线性基因组的序列预测-70蛋白质编码基因（84）。进入HSV DNA之后 培养细胞的细胞核，一系列基因表达[除非另有引用，否则请参见（97）]。 一类基因的转录（即立即提早，即a）被病毒座刺激 成分VP16，并且不需要先前的病毒蛋白合成。 IE基因产品包括ICP 4、27、0， 22是基因表达的调节剂和ICP47，它拮抗抗原表现。 ICP4 27对于病毒复制至关重要。 ICPO，ICP22和ICP47是非必需的。但是，ICPO是 在某些细胞中，在静息细胞和ICP22的低多重感染中尤其重要 类型比其他类型。随后要表达的两类基因被称为早期（E，（3）及以后 （l，y）分别基于在感染周期中其表达峰的何时。 E基因主要编码 参与DNA复制的蛋白质。这些包括直接参与DNA合成的酶， PHS 398/2590（修订版09/04）页面延续格式页面 000137