SARS-CoV-2 Nsp1 N-terminal and linker regions as a platform for host translational shutoff_CLMS_DSSO_dataset

In the early stages of SARS-CoV-2 infection, non-structural protein 1 (Nsp1) inhibits the innate immune response by inserting its C-terminal helices into the mRNA entry channel of the ribosome and promoting mRNA degradation. However, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains unclear. As the conserved N-terminal domain (NTD) and linker region has been implicated with this process, we set out to characterize the interactome of Nsp1 and its topology by crosslinking mass spectrometry. The distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit. We find that Nsp1 is in contact with 40S proteins lining the mRNA entry channel and the G subunit of the eIF3 complex. We further show that Nsp1 predominantly binds to initiating ribosomes by affinity purification mass spectrometry and sucrose gradient ultracentrifugation. Our results point towards a potential mechanism by which Nsp1 is preferentially recruited to host-translating ribosomes, leading to selective inhibition of canonical initiation intermediates and subsequent mRNA degradation.