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

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. Nevertheless, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains elusive. We set out to characterize the interactome of full-length Nsp1 and its topology by crosslinking mass spectrometry in order to investigate the role of the N-terminal domain and linker regions in host translational shutoff. We find that these regions are in contact with 40S proteins lining the mRNA entry channel and detect a novel interaction with the G subunit of the eIF3 complex. The crosslink-derived distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit, reporting on the dynamic interface between Nsp1, the ribosome and the eIF3 complex. The significance of the Nsp1-eIF3G interaction is supported by further evidence that Nsp1 predominantly binds to 40-43S complexes. Our results point towards a mechanism by which Nsp1 is preferentially recruited to canonical initiation complexes, leading to selective inhibition of host-translating ribosomes and subsequent mRNA degradation. ### Competing Interest Statement The authors have declared no competing interest.