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

甲型病毒是病媒传播的病原体，已被证明能够导致临床疾病的重大爆发。目前，绝对缺乏安全和经批准的抗病毒疗法/疫苗。因此，确定感染的分子基础的研究不仅对于理解甲型病毒的发病机制，而且对于开发创新的抗病毒策略，都是至关重要的。这项建议的主要研究目标是确定一种新的甲型病毒RNA物种影响感染和发病的机制。支持这一目标的科学前提和理论基础基于大量的初步证据，这些证据表明，非封顶基因组RNA(NcgRNAs)的产生是甲病毒感染所必需的，涉及辛德比斯病毒(SINV)和罗斯河病毒(Ross River Virus)的研究证明了这一点。尽管如此，ncgRNAs对病毒感染的影响程度尚不清楚，这是知识库中的一个严重缺口。因此，我们提出了以下具体目标：1)确定非封顶基因组vRNA影响感染的机制；2)确定非封顶基因组vRNA对病毒致病的影响。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ncgRNAs影响病毒感染的机制。具体地说，我们将通过一种基于IRES的新方法来确定ncgRNA功能是否与其可译性有关。此外，我们将确定哪个病毒过程受到ncgRNA还原的负面影响；并确定ncgRNA功能所需的分子相互作用。第二个目的是采用体内研究和干扰素活性组织培养模型相结合的方法，全面确定ncgRNAs在SINV致病中的作用。简而言之，ncgRNAs在诱导有害的神经炎性反应中的作用将在组织学、转录组和蛋白质组水平进行深入的评估。此外，在高度可处理的组织培养感染模型中，增加和降低甲型病毒封闭率对宿主先天免疫反应的影响也将被定义。这项拟议的工作具有非常重要的意义，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，拟议的研究在概念和技术上都是创新的，因为最新的感染技术、方法和模型被用来描绘新的ncgRNA物种在分子感染和发病机制中的贡献。这项工作的成功完成将消除上述理解方面的关键差距，从而从根本上扩大知识库；此外，拟议的研究很有可能导致确定新的抗病毒策略。

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.