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: 8602830
• 负责人: David C. Bloom
• 金额: $ 37.5万
• 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/8602830
• 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.

描述（由申请人提供）：1型单纯疱疹病毒（HSV-1）通常感染口腔粘膜，并在感觉神经元内建立终身潜伏感染。在潜伏期，病毒裂解基因被抑制，只有一种转录物大量转录，即潜伏相关转录物（LAT）。HSV-1潜伏期的特征是间歇性复发，在此期间，存在于一些神经元中的病毒基因组重新激活。病毒颗粒产生并被运送到感染的原始部位，导致临床疾病。因此，HSV-1是导致显著发病率的原因，并且是美国感染性失明的主要原因。虽然抗病毒药物可以降低复发的严重程度，但没有治愈方法。显然，了解HSV如何调节感染的裂解期和潜伏期的分子基础可以提供新的治疗方法。 最近，8种microRNA（miRNAs）被证明在HSV-1 LAT区域内或附近编码。体外分析表明，这些miRNA中至少有两种调节HSV-1 IE基因表达。该研究旨在确定这些miRNAs在体内HSV-1感染的病理生物学中发挥的作用。为了实现这一目标，我们将：1）构建在8种HSV-1 miRNA中含有失活突变的HSV-1重组体，并评估这些突变病毒的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体的复制、传播、潜伏期的建立和再活化能力的变化。将拯救表现出改变的表型的突变体，并研究表型变化的分子基础; 3）使用强大的光活化核糖核苷增强的交联和免疫沉淀（PAR-CLIP）技术来直接鉴定用野生型HSV-1或用缺乏特异性miRNA的HSV-1变体感染的细胞中HSV-1 miRNA占据的mRNA靶位点。通过这些分析鉴定的病毒或细胞mRNA靶标将通过QT-RTPCR进行确认，随后通过敲低和过表达分析（如适用）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，拟议的项目汇集了布鲁姆和卡伦实验室的综合专业知识。佛罗里达大学的Bloom实验室在HSV-1重组体的构建和表征以及HSV-1发病机制的研究方面具有专业知识，包括小鼠和兔模型中的潜伏期和再激活。杜克大学的Cullen实验室鉴定了HSV-1 LAT区域内编码的miRNA，并具有使用PAR-CLIP和其他测定法分析miRNA表达和功能的广泛专业知识。总的来说，所提出的表征HSV-1 LAT miRNAs功能的综合方法应该为调节HSV-1感染的裂解和潜伏期的分子过程提供关键见解，并为miRNAs如何调节疱疹病毒家族成员提供新的范例。