Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry (RAP-MS), we recently characterized the first atlas of human and viral proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We now expand these results to different cell types and resolve the interactomes of genomic and subgenomic viral RNAs separately. We report a subgenome-resolved network of host RNA binders and find quantitative differences in the enrichment of host factors towards different viral RNA types, indicative of distinct binding preferences within the SARS-CoV-2 RNA-protein interactome. We globally map direct interaction sites of viral and host cell proteins on SARS-CoV-2 RNAs and use genetic perturbation together with pharmacological inhibition to demonstrate the functional relevance of several direct RNA binders in SARS-CoV-2 infections. The identification of host dependency factors and defense strategies as presented here provides a general roadmap for dissecting the biology of RNA viruses and the interactions between hosts and pathogens at the molecular level with therapeutic implications.
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