Arthropod-borne viruses, such as the members of the genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish and complete their life cycles. Despite considerable efforts to define the host-pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA-protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduce a robust viral RNA-protein discovery method to elucidate the Sindbis virus (SINV) RNA-protein host interface. Cross-link-assisted mRNP purification (CLAMP) assessment revealed an extensive array of host-pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of protein-vRNA interaction by a UV cross-linking and immunoprecipitation sequencing (CLIP-seq) approach and assessed the consequences of the RNA-protein binding event of hnRNP K, hnRNP I, and hnRNP M in regard to viral infection. Here, we demonstrate that mutation of the prioritized hnRNP-vRNA interaction sites effectively disrupts hnRNP-vRNA interaction. Correlating with disrupted hnRNP-vRNA binding, SINV growth kinetics were reduced relative to wild-type parental viral infections in vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics was found to be dysregulated structural-gene expression. Collectively, this study further defines the scope and importance of the alphavirus host-pathogen vRNA-protein interactions.

IMPORTANCE Members of the genus Alphavirus are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their life cycle. However, the extent and importance of host-pathogen RNA-protein interactions are woefully undercharacterized. The efforts detailed in this study fill this critical gap, and the significance of this research is 3-fold. First, the data presented here fundamentally expand the scope and understanding of alphavirus host-pathogen interactions. Second, this study identifies the sites of interaction for several prioritized interactions and defines the contribution of the RNA-protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of the given host-pathogen interactions may be assessed in the future, using a mouse model of infection.

节肢动物传播的病毒，例如甲病毒属的成员是全球公共卫生的重要关注点。作为专一的细胞内病原体，RNA病毒必须与宿主细胞机制相互作用，以建立并完成其生命周期。尽管为定义α病毒复制所必需的宿主-病原体相互作用付出了巨大的努力，但尚未对α病毒RNA-蛋白质相互作用进行公正且包容的评估。而且，在它们作为RNA结合蛋白的分子功能的全部范围内，这些相互作用的生物学和分子重要性还没有被完全认识到。此处提供的数据介绍了一种鲁棒的病毒RNA蛋白发现方法，用于阐明Sindbis病毒（SINV）RNA蛋白宿主接口。交联辅助mRNP纯化（CLAMP）评估揭示了以病毒RNA（vRNA）为中心的大量宿主-病原体相互作用。在对与vRNA相关的宿主蛋白进行优先排序后，我们通过UV交联和免疫沉淀测序（CLIP-seq）方法确定了蛋白-vRNA相互作用的位点，并评估了hnRNP K的RNA-蛋白结合事件的后果，关于病毒感染的hnRNP I和hnRNPM。在这里，我们证明了优先hnRNP-vRNA相互作用位点的突变有效地破坏了hnRNP-vRNA相互作用。与破坏的hnRNP-vRNA结合相关，在感染的脊椎动物和无脊椎动物组织培养模型中，相对于野生型亲本病毒感染，SINV生长动力学降低。发现导致病毒生长动力学降低的分子机制是结构基因表达失调。总体而言，这项研究进一步定义了甲病毒宿主-病原体vRNA-蛋白质相互作用的范围和重要性。

重要Alphavirus属的成员因其可能导致严重疾病而广为人知。尽管有这种认识，但目前尚无抗病毒疗法或安全有效的疫苗可用于治疗α病毒感染。甲病毒利用宿主细胞机制有效地建立并完成其生命周期。但是，宿主-病原体RNA-蛋白质相互作用的程度和重要性严重不足。这项研究中详细介绍的工作填补了这一关键空白，这项研究的意义是三倍的。首先，此处提供的数据从根本上扩展了α病毒宿主与病原体相互作用的范围和理解。其次，这项研究确定了几种优先相互作用的相互作用位点，并确定了RNA-蛋白质相互作用在分子水平上的作用。最后，