Building a recombinant Herpesvirus core laboratory to systematically analyze the

描述（由申请方提供）：与疱疹病毒建立持续性和潜伏性感染的能力一致，所有疱疹病毒基因中约25%调节和/或消除宿主细胞对病毒感染的反应。最近，已经在a-、B-和g-疱疹病毒基因组中鉴定了140种microRNA，其对于CMV和KSHV也靶向宿主免疫功能。MicroRNA是短的22 + 3 nt RNA，其通过结合mRNA的3 'UTR并诱导翻译沉默或RNA降解来转录后调节基因表达。到目前为止，只有少数基因被实验证明是miRNA靶点。从这些有限的研究中可以清楚地看出，病毒miRNA调节基本的细胞过程，包括先天性和适应性免疫、血管生成和细胞凋亡，以及疱疹病毒生命周期中的关键步骤、潜伏期和从潜伏复制到裂解复制的转换。microRNA对这些过程的调节可能会影响宿主/病毒相互作用，从而直接导致病毒发病。此外，了解miRNAs如何靶向先天性和适应性免疫功能对于疱疹病毒疫苗的开发至关重要。为了研究人类疱疹病毒编码的miRNA的作用，我们建议建立一个核心实验室，其使命是系统地产生携带单个和多个miRNA突变的疱疹病毒基因组文库。为了消除二次突变的可能性，将使用我们完善的大规模平行测序设施对重组基因组进行重新测序。这种共同努力将创造出非常有价值的试剂，从而能够在动物模型的背景下研究miRNA在病毒感染背景下如何发挥作用，以及在适当的系统存在的情况下。重要的是，经过质量控制后，每一种突变病毒都将免费与研究界分享。为这一专门任务创建一个核心实验室将产生很大的影响，并显着增加在这一新颖和高度重要的调查领域的研究成果。此外，在未来，这样的核心实验室可以扩大到其他基因的靶向诱变，可以要求在任何美国研究地点的研究人员在收费的基础上。 公共卫生相关性：最近，在与癌症相关的人类病毒中发现了一类新的非编码RNA（microRNA）。对它们功能的初步了解表明它们在免疫逃避和发病机制中的重要作用，就像它们在包括许多癌症在内的疾病中所涉及的人类对应物一样。该提案旨在建立一个服务实验室，该实验室将创建在这些microRNA基因中具有特定突变的病毒，这是一项艰巨且具有技术挑战性的任务。新成立的实验室将雇用五名科学家，然后将向该领域的所有研究人员提供这些有价值的试剂。因此，这一项目将大大加快缩小这一知识差距的进展，这对人类疾病非常重要。

DESCRIPTION (provided by applicant): Consistent with the ability of herpesviruses to establish persistent and latent infections, about 25% of all herpesvirus genes modulate and/or abrogate host cellular responses to viral infection. Recently, 140 microRNAs have been identified in a-, b-, and g-herpesvirus genomes which for CMV and KSHV also target host immune functions. MicroRNAs are short 22 + 3 nt RNAs that post-transcriptionally regulate gene expression by binding to 3'UTRs of mRNAs and inducing translational silencing or RNA degradation. To date, only a few genes have been experimentally proven to be miRNA targets. From these limited studies it is clear that viral miRNAs regulate fundamental cellular processes including innate and adaptive immunity, angiogenesis, and apoptosis, and key steps in the herpesvirus life cycle, latency and the switch from latent to lytic replication. Modulation of these processes by microRNAs is likely to affect host/virus interactions and thereby directly contribute to viral pathogenesis. In addition, understanding how miRNAs target innate and adaptive immune function will be critically important for herpesvirus vaccine development. To study the role of human herpesvirus-encoded miRNAs, we propose to establish a core laboratory with the mission to systematically generate a library of herpesvirus genomes that carry single and multiple miRNA mutations. To eliminate the possibility of secondary mutations, recombinant genomes will be re-sequenced using our well established massively parallel sequencing facility. This concerted effort will create highly valuable reagents that will enable studies on how miRNAs function in the context of viral infection, and where appropriate systems exist, in the context of animal models. Importantly, after quality control, each mutant virus will be freely shared with the research community. Creating a core laboratory for this specialized task will have high impact and significantly increase research output in this novel and highly significant field of investigation. Furthermore, in the future, such a core laboratory can be expanded towards targeted mutagenesis of additional genes that can be requested on a for fee basis by investigators at any US research site. PUBLIC HEALTH RELEVANCE: Recently, a novel class of non-coding RNAs (microRNAs) has been identified in human viruses associated with cancer. First insights into their function suggest important roles in immune evasion and pathogenesis like their human counterparts which are involved in diseases including many cancers. This proposal aims to establish a service laboratory that would create viruses with specific mutations in these microRNA genes, a task which is laborious and technically challenging. The newly created laboratory, which will employ five scientists, will then provide these valuable reagents to all investigators in the field. As a result, this project would dramatically accelerate progress towards closing this knowledge gap, which is highly significant for human disease.