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

ABSTRACT: 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.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Juri Rappsilber

PROVIDER: PXD031688 | JPOST Repository | Wed Feb 15 00:00:00 GMT 2023

REPOSITORIES: jPOST

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			Action	DRS
	B210515_02_HF_AG_IN_120_ID_Nsp1A_Ribofract_5_inj2.raw	Raw
	B210515_04_HF_AG_IN_120_ID_Nsp1A_Ribofract_6_inj2.raw	Raw
	B210515_06_HF_AG_IN_120_ID_Nsp1A_Ribofract_7_inj2.raw	Raw
	B210515_08_HF_AG_IN_120_ID_Nsp1A_Ribofract_8.raw	Raw
	B210515_10_HF_AG_IN_120_ID_Nsp1A_Ribofract_9.raw	Raw

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Project description: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.

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