Regulation of lytic and latent infection by HSV-1 encoded miRNAs

HSV-1 编码的 miRNA 对裂解和潜伏感染的调节

• 批准号: 8219674
• 负责人: David C. Bloom
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8219674
• 关键词: Acute; Affect; Afferent Neurons; Antiviral Agents; Binding; Biological Assay; Blindness; Cells; Clinical; Deletion Mutation; Development; Disease; Encephalitis; Episome; Epithelial; Family member; Gene Expression; Gene Silencing; Genes; Genome; Goals; Herpes Labialis; Herpesviridae; Herpesvirus 1; Human; Immunoprecipitation; In Vitro; Individual; Infection; Invaded; Investigation; Laboratories; Lead; Life; Lytic; Lytic Phase; Messenger RNA; MicroRNAs; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Nerve Tissue; Neurons; Oral mucous membrane structure; Oryctolagus cuniculus; Pathogenesis; Phase; Phenotype; Play; Process; Recombinants; Recurrence; Regulation; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Ribonucleosides; Role; Severities; Simplexvirus; Site; Spinal Ganglia; System; Techniques; Testing; Tissues; Transcript; Undifferentiated; Variant; Viral; Viral Genes; Viral Genome; Virion; Virulent; Virus; Virus Diseases; Virus Latency; Western Blotting; Work; base; cellular engineering; crosslink; defined contribution; design; in vivo; insight; interest; knock-down; latency associated transcript; latent infection; lytic replication; mutant; neurovirulence; novel; novel therapeutic intervention; overexpression; reactivation from latency; relating to nervous system

DESCRIPTION (provided by applicant): Herpes simplex virus type 1 (HSV-1) typically infects the oral mucosa and establishes a life-long latent infection within sensory neurons. During latency, the viral lytic genes are repressed and only one transcript is abundantly transcribed, the latency associated transcript (LAT). HSV-1 latency is characterized by intermittent episodes of recurrence during which the viral genomes present in some neurons reactivate. Virus particles are produced and transported to the original site of infection, resulting in clinical disease. Consequently, HSV-1 is responsible for significant morbidity and is the leading cause of infectious blindness in the US. While antivirals can reduce the severity of the recurrences, there is no cure. Clearly, understanding the molecular basis of how HSV regulates the lytic and latent phases of infection could provide new therapeutic approaches. Recently 8 microRNAs (miRNAs) were shown to be encoded within and adjacent to the HSV-1 LAT region. In vitro analyses have demonstrated that at least two of these miRNAs regulate HSV-1 IE gene expression. The proposed study aims to determine the roles that these miRNAs play in the pathobiology of HSV-1 infections in vivo. In order to accomplish this goal we will: 1) Construct HSV-1 recombinants containing inactivating mutations in the 8 HSV-1 miRNAs and assess these mutant viruses for changes in viral gene expression and replication in vitro; 2) Analyze these recombinants in the mouse and rabbit models for changes in replication, spread, establishment of latency and ability to reactivate. Recombinants that display altered phenotypes will be rescued and the molecular basis of the phenotypic change investigated; 3) Use the powerful photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) technique to directly identify mRNA target sites occupied by HSV-1 miRNAs in cells infected with wild-type HSV-1 or with HSV-1 variants lacking specific miRNAs. Viral or cellular mRNA targets identified by these analyses will be confirmed through QT-RTPCR followed by investigation of the functional role of that gene during the viral infection cycle by knock-down and over-expression analyses, where appropriate. In order to accomplish these goals the proposed project brings together the combined expertise of the Bloom and Cullen labs. The Bloom lab at UF has expertise in the construction and characterization of HSV-1 recombinants and the study of HSV-1 pathogenesis, including latency and reactivation in the mouse and rabbit models. The Cullen lab at Duke identified the miRNAs encoded within the HSV-1 LAT region and has extensive expertise analyzing miRNA expression and function using PAR-CLIP and other assays. In total, the proposed comprehensive approach to characterize the function of the HSV-1 LAT miRNAs should provide key insights into the molecular processes that regulate the lytic and latent phases of HSV-1 infection and provide new paradigms of how miRNAs regulate herpesvirus family members in general. PUBLIC HEALTH RELEVANCE: Herpes simplex virus type 1 (HSV-1) causes cold sores and other serious disease in humans, including encephalitis and blindness, and while there are antiviral drugs to treat herpes they don't completely block the infection and there is no cure. The goal of the proposed study is to determine if HSV-1 encoded microRNAs (miRNAs) control the ability of HSV-1 to establish latency and/or reactivate from latency to cause disease. Clearly defining the contribution of viral miRNAs to HSV-1-induced disease, and defining the mRNAs that these miRNAs regulate, could lead to the development of better therapies for treating herpes infections in humans.

描述（由申请人提供）：单纯疱疹病毒类型1（HSV-1）通常会感染口腔粘膜，并在感觉神经元内建立终身潜伏感染。在潜伏期期间，病毒裂解基因被抑制，只有一个转录本被录制，延迟相关转录本（LAT）。 HSV-1潜伏期的特征是复发的间歇发作，在此期间，某些神经元中存在病毒基因组会重新激活。生产病毒颗粒并将其运输到原始感染部位，从而导致临床疾病。因此，HSV-1负责明显的发病率，是美国传染失明的主要原因。尽管抗病毒药可以减少复发的严重程度，但无法治愈。显然，了解HSV如何调节感染的裂解和潜在阶段的分子基础可以提供新的治疗方法。 最近，显示了8个microRNA（miRNA）在HSV-1 LAT区域内和附近编码。体外分析表明，这些miRNA中至少有两个调节HSV-1 IE基因表达。拟议的研究旨在确定这些miRNA在体内HSV-1感染的病理生物学中所起的作用。为了实现这一目标，我们将：1）在8 HSV-1 miRNA中构建含有灭活突变的HSV-1重组者，并评估这些突变病毒，以了解病毒基因表达和体外复制的变化； 2）在小鼠和兔模型中分析这些重组因素，以改变复制，扩散，延迟的建立和重新激活的能力。表现出改变表型的重组将被救出，并研究了表型变化的分子基础； 3）使用强大的光活化核糖核苷增强交联和免疫沉淀（PAR-CLIP）技术直接鉴定HSV-1 miRNA占据的mRNA靶位点在感染了野生型HSV-1的细胞中或缺乏HSV-1变体的野生型HSV-1或缺乏特定特定miRNA的细胞。这些分析确定的病毒或细胞mRNA靶标将通过QT-RTPCR确认，然后通过合适的敲低和过表达分析来研究该基因在病毒感染周期中的功能作用。 为了实现这些目标，拟议的项目汇集了Bloom和Cullen Labs的综合专业知识。 UF的Bloom Lab在HSV-1重组剂的构建和表征以及HSV-1发病机理的研究中具有专业知识，包括小鼠和兔模型中的潜伏和重新激活。杜克（Duke）的库伦（Cullen）实验室确定了在HSV-1 LAT区域中编码的miRNA，并具有广泛的专业知识，可以使用PAR-CLIP和其他测定法分析miRNA的表达和功能。总的来说，提出的综合方法来表征HSV-1 LAT miRNA的功能，应提供对调节HSV-1感染的裂解和潜在阶段的分子过程的关键见解，并提供MiRNA如何调节疱疹病毒家族成员的新范式。 公共卫生相关性：单纯疱疹病毒1型（HSV-1）会引起人类的唇疱疹和其他严重疾病，包括脑炎和失明，虽然有抗病毒药物可以治疗疱疹，但它们并没有完全阻止感染，并且无法治愈。拟议的研究的目的是确定HSV-1编码的microRNA（miRNA）是否控制HSV-1建立潜伏期和/或从潜伏期引起疾病的能力。清楚地定义了病毒miRNA对HSV-1诱导的疾病的贡献，并定义了这些miRNA调节的mRNA，可能会导致开发更好的治疗人类疱疹感染的疗法。