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.