Building a recombinant Herpesvirus core laboratory to systematically analyze the

描述(申请人提供)：与疱疹病毒建立持续和潜伏感染的能力一致，大约25%的疱疹病毒基因调节和/或消除宿主细胞对病毒感染的反应。最近，在a-、b-和g-疱疹病毒基因组中发现了140个microRNAs，它们也针对CMV和KSHV的宿主免疫功能。MicroRNAs是一种短小的22+3ntRNA，它通过与mRNAs的3‘端非编码区结合，诱导翻译沉默或RNA降解，在转录后调节基因的表达。到目前为止，只有几个基因被实验证明是miRNA的靶标。从这些有限的研究中可以清楚地看出，病毒miRNAs调控基本的细胞过程，包括先天性和获得性免疫、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。MicroRNAs对这些过程的调控可能会影响宿主/病毒的相互作用，从而直接促进病毒的致病。此外，了解miRNAs如何针对先天性和获得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miRNAs的作用，我们建议建立一个核心实验室，其任务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，重组基因组将使用我们成熟的大规模并行测序设施进行重新测序。这种协调一致的努力将创造出非常有价值的试剂，使人们能够在病毒感染的背景下研究miRNAs如何发挥作用，并在动物模型的背景下研究适当的系统。重要的是，在质量控制之后，每一种变异病毒都将与研究社区自由分享。为这一专门任务创建一个核心实验室将产生很大影响，并显著增加这一新的和非常重要的调查领域的研究产出。此外，在未来，这样的核心实验室可以扩展到对更多基因进行定向突变，这些基因可以由美国任何研究地点的研究人员付费要求。 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(MicroRNAs)。首先，对它们功能的洞察表明，它们在免疫逃避和发病中扮演着重要角色，就像它们在包括许多癌症在内的疾病中所涉及的人类同行一样。这项提议旨在建立一个服务实验室，创造具有这些microRNA基因特定突变的病毒，这是一项艰巨的任务，在技术上具有挑战性。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，该项目将极大地加快缩小这一知识鸿沟的进展，这对人类疾病具有重要意义。

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.