Comprehensive Definition of the Critical Role(s) of the Alphaviral Noncapped Genomic RNAs to Infection and Pathogenesis

甲病毒是媒介传播的病原体，已证明能够引起临床疾病的重大爆发。目前，绝对缺乏安全和批准的抗病毒疗法/疫苗。因此，定义感染的分子基础的研究不仅对理解甲病毒的发病机制至关重要，而且对开发创新的抗病毒策略至关重要。该提案的总体研究目标是确定新型甲病毒RNA种类影响感染和发病机制的机制。支持这一目标的科学前提和基本原理是基于大量的初步证据，这些证据表明，无帽基因组RNA（ncgRNA）的产生对于甲病毒感染至关重要，如涉及辛德毕斯病毒（SINV）和罗斯河病毒的研究所证明的那样。尽管如此，ncgRNA影响病毒感染的程度是未知的，代表了知识库中的关键空白。因此，我们提出了以下具体目标- i）确定无帽基因组vRNA影响感染的机制，和ii）确定无帽基因组vRNA对病毒发病机制的影响。为了实现第一个目标，我们将对SINV感染进行详尽的分子分析，以确定ncgRNA影响病毒感染的机制。具体来说，我们将通过一种新的基于IRES的方法来确定ncgRNA功能是否与其可翻译性有关。此外，我们将确定哪些病毒过程受到ncgRNA减少的负面影响;并确定ncgRNA功能所需的分子相互作用。第二个目标使用体内研究和IFN活性组织培养模型的组合方法来全面定义ncgRNA对SINV发病机制的贡献。简而言之，将在组织学上以及在转录组和蛋白质组水平上深入评价ncgRNA对诱导有害神经炎症反应的贡献。此外，增加和降低甲病毒加帽效率对宿主先天免疫应答的影响也将在高度易处理的感染组织培养模型中定义。拟议的工作是非常重要的，因为它试图表征和定义一种新的病毒RNA物种的分子重要性。此外，所提出的研究在概念上和技术上都是创新的，因为最先进的技术、方法和感染模型被用于描述新的ncgRNA种类对分子感染和发病机制的贡献。这项工作的成功完成将从根本上扩大知识库，消除上述理解的关键差距;此外，拟议的研究很有可能导致识别新的抗病毒策略。

Alphaviruses are vector-borne pathogens that have proven capable of causing significant outbreaks of clinical disease. Currently, there is an absolute lack of safe and approved antiviral therapies / vaccines. Thus, research which defines the molecular basis of infection is essential and critically important to not only understanding alphaviral pathogenesis, but to the development of innovative antiviral strategies. The overarching research objective of this proposal is to define the mechanism by which a novel alphavirus RNA species impacts infection and pathogenesis. The scientific premise and rationale supporting this objective are based upon a wealth of preliminary evidence which indicates that the production of noncapped genomic RNAs (ncgRNAs) is essential for alphaviral infection, as evidenced by studies involving Sindbis virus (SINV) and Ross River virus. Nonetheless, the extent to which the ncgRNAs impact viral infection is unknown and represents a critical gap in the knowledge base. Thus, we propose the following specific aims- i) To determine the mechanism with which the noncapped genomic vRNAs impact infection, and ii) To define the impact of the noncapped genomic vRNAs on viral pathogenesis. To accomplish the first aim, we will undertake an exhaustive molecular analysis of SINV infection to determine the mechanism by which the ncgRNAs effect viral infection. Specifically, we will determine whether or not ncgRNA function is linked to its translatability via a novel IRES-based approach. In addition, we will determine which viral process is negatively affected by ncgRNA reduction; and determine the molecular interactions required for ncgRNA function. The second aim uses a combinatorial approach of in vivo studies and IFN-competent tissue culture models to comprehensively define the contribution of the ncgRNAs to SINV pathogenesis. Briefly, the contribution of the ncgRNAs to the induction of a detrimental neuroinflammatory response will be evaluated in depth histologically, and at the transcriptomic and proteomic levels. Moreover, the consequences of increasing and decreasing alphaviral capping efficiency on the host innate immune response will also be defined in highly tractable tissue culture models of infection. The proposed work is highly significant in that it seeks to characterize and define the molecular importance of a novel viral RNA species. Moreover, the proposed research is conceptually and technically innovative as state-of-the-art techniques, approaches, and models of infection are used to delineate the contributions of the novel ncgRNA species to molecular infection and pathogenesis. The successful completion of this work will fundamentally expand the knowledgebase by eliminating the critical gap in understanding described above; in addition, the proposed research has a high likelihood of leading to the identification of novel antiviral strategies.