SARS-CoV-2 Nsp1 cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA

A better mechanistic understanding of virus-host dependencies can help reveal vulnerabilities and identify opportunities for therapeutic intervention. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in protein synthesis dynamics during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones that are used by CoV2 to promote the synthesis and maturation of its own proteins. These can be targeted to inhibit infection, without major toxicity to the host. We also find that CoV2 non-structural protein 1 (Nsp1) cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA. When EIF1/1A are depleted, more ribosomes initiate translation from a conserved upstream CUG start codon found in all genomic and subgenomic viral RNAs. This results in higher translation of an upstream open reading frame (uORF1) and lower translation of the main ORF, altering the stoichiometry of viral proteins and attenuating infection. Replacing the upstream CUG with AUG strongly inhibits translation of the main ORF independently of Nsp1, EIF1, or EIF1A. Taken together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and proposes a model for dosage control of viral proteins through Nsp1-mediated control of translation start site selection. Host-directed antivirals offer a promising therapeutic approach for many viruses, including SARS-CoV-2 (CoV2), but their development requires a deeper understanding of virus-host interactions. Of particular interest are interactions that selectively promote the synthesis of viral proteins, including RNA-binding proteins, translation factors and molecular chaperones. Drugs that inhibit such biosynthetic factors have already entered clinical trials for multiple indications. To identify new cellular targets for intervention, we isolated translating ribosomes from CoV2-infected and control cells and analyzed their interacting partners by mass-spectrometry. We found multiple biosynthetic factors that were specifically enriched on polysomes translating CoV2, including translation initiation factors EIF1 and 1A. These factors control translation start site selection, cooperate with the viral non-structural protein 1 (Nsp1) to selectively enhance translation of viral genomic RNA, and are exploited by CoV2 to regulate the timing and stoichiometry of viral protein synthesis. Targeting EIF1A by siRNA reduces infection with minimal toxicity to the host. Although the nature of interactions between EIF1/1A and Nsp1 remains unclear, this interdependency may provide a new strategy for antiviral therapy.