Building a recombinant Herpesvirus core laboratory to systematically analyze the

描述（由申请人提供）：与疱疹病毒建立持续性和潜伏性感染的能力一致，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microrna， CMV和KSHV也靶向宿主免疫功能。MicroRNAs是一种短的22 + 3 nt RNA，通过结合mrna的3' utr诱导翻译沉默或RNA降解来转录后调节基因表达。迄今为止，只有少数基因被实验证明是miRNA的靶标。从这些有限的研究中可以清楚地看出，病毒mirna调节基本的细胞过程，包括先天和适应性免疫、血管生成和细胞凋亡，以及疱疹病毒生命周期、潜伏期和从潜伏到裂解复制的转换的关键步骤。microrna对这些过程的调节可能会影响宿主/病毒的相互作用，从而直接影响病毒的发病机制。此外，了解mirna如何靶向先天和适应性免疫功能对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，其任务是系统地生成携带单个和多个miRNA突变的疱疹病毒基因组库。为了消除二次突变的可能性，重组基因组将使用我们完善的大规模平行测序设备进行重新测序。这一共同努力将创造出非常有价值的试剂，使研究mirna在病毒感染背景下的功能，以及在动物模型中存在适当系统的情况下。重要的是，经过质量控制后，每个突变病毒将与研究界免费共享。为这一专业任务创建一个核心实验室将在这一新颖而高度重要的研究领域产生重大影响并显著增加研究成果。此外，在未来，这样一个核心实验室可以扩展到其他基因的靶向诱变，这些基因可以由美国任何研究地点的研究人员收费要求。

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.