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

描述(申请人提供)：单纯疱疹病毒1型(HSV-1)典型地感染口腔粘膜，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，只有一个转录本被大量转录，即潜伏期相关转录本(LAT)。HSV-1潜伏期的特点是间歇性复发，在此期间存在于一些神经元中的病毒基因组重新激活。病毒颗粒被产生并运输到最初的感染部位，导致临床疾病。因此，HSV-1是导致严重发病率的原因，也是美国传染性失明的主要原因。虽然抗病毒药物可以减轻复发的严重程度，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个microRNAs(MiRNAs)被发现编码在HSV-1LAT区域内和邻近区域。体外分析表明，这些miRNAs中至少有两个调控HSV-1 IE基因的表达。这项拟议的研究旨在确定这些miRNAs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)构建包含8个HSV-1 miRNAs失活突变的HSV-1重组体，并评估这些突变病毒在体外病毒基因表达和复制的变化；2)在小鼠和兔模型中分析这些重组体在复制、传播、潜伏期的建立和重新激活能力方面的变化。表型改变的重组体将被拯救，并研究表型改变的分子基础；3)使用强大的可光激活的核糖核苷增强的交联和免疫沉淀(PAR-CLIP)技术，在感染野生型HSV-1或缺乏特异性miRNAs的HSV-1变异体感染细胞中，直接确定HSV-1 miRNAs占据的mRNA靶点。通过这些分析确定的病毒或细胞信使核糖核酸目标将通过Qt-RTPCR进行确认，然后在适当的情况下通过敲除和过度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，拟议的项目汇集了布卢姆和库伦实验室的综合专业知识。UF的Bloom实验室在HSV-1重组体的构建和特性以及HSV-1致病机制的研究方面拥有专业知识，包括小鼠和兔模型中的潜伏期和重新激活。杜克大学的库伦实验室确定了HSV-1LAT区域内编码的miRNAs，并拥有使用PAR-CLIP和其他分析方法分析miRNA表达和功能的丰富专业知识。总之，提出的表征HSV-1 LAT miRNAs功能的综合方法应该为调节HSV-1感染的裂解期和潜伏期的分子过程提供关键的见解，并为miRNAs如何调控疱疹病毒家族成员提供新的范例。 与公共卫生相关：1型单纯疱疹病毒(HSV-1)会导致人类冻疮和其他严重疾病，包括脑炎和失明，虽然有治疗疱疹的抗病毒药物，但它们不能完全阻止感染，也没有治愈方法。这项拟议研究的目的是确定HSV-1编码的microRNAs(MiRNAs)是否控制HSV-1建立潜伏期和/或从潜伏期重新激活导致疾病的能力。明确定义病毒miRNAs在HSV-1诱导的疾病中的作用，并定义这些miRNAs调节的mRNAs，可能会导致开发出更好的治疗人类疱疹感染的方法。

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.