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

描述（由申请人提供）：1型单纯疱疹病毒（HSV-1）通常感染口腔黏膜，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，只有一种转录物被大量转录，即潜伏期相关转录物（LAT）。1型单纯疱疹病毒潜伏期的特点是间歇性复发，在此期间，存在于某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，1型单纯疱疹病毒是导致显著发病率的原因，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，但无法治愈。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。最近发现有8个microrna （mirna）编码在HSV-1 LAT区域内或附近。体外分析表明，这些mirna中至少有两种调节HSV-1 IE基因的表达。该研究旨在确定这些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)构建含有8个HSV-1 mirna失活突变的HSV-1重组病毒，并评估这些突变病毒在体外的病毒基因表达和复制变化；2)在小鼠和家兔模型中分析这些重组体在复制、传播、潜伏期建立和再激活能力方面的变化。显示表型改变的重组体将被拯救，表型变化的分子基础将被调查；3)利用强大的光激活核糖核苷增强交联和免疫沉淀（PAR-CLIP）技术，在感染野生型HSV-1或缺乏特异性miRNAs的HSV-1变体的细胞中，直接鉴定HSV-1 miRNAs所占的mRNA靶位点。通过这些分析确定的病毒或细胞mRNA靶标将通过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实验室的专业知识。佛罗里达大学的Bloom实验室在HSV-1重组的构建和表征以及HSV-1发病机制的研究方面具有专长，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区域编码的miRNA，并具有使用PAR-CLIP和其他检测分析miRNA表达和功能的广泛专业知识。总之，所提出的描述HSV-1 LAT mirna功能的综合方法将为了解调节HSV-1感染的裂解期和潜伏期的分子过程提供关键见解，并为mirna如何调节疱疹病毒家族成员提供新的范例。

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.