In vitro reconstitution of SARS-CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on β-globin, EMCV IR...

Full description

Saved in:
Bibliographic Details
Published in:Genes & development 2023-09, Vol.37 (17-18), p.844-860
Main Authors: Abaeva, Irina S, Arhab, Yani, Miścicka, Anna, Hellen, Christopher U T, Pestova, Tatyana V
Format: Article
Language:English
Subjects:
COVID-19
Humans
Peptide Initiation Factors - genetics
Peptide Initiation Factors - metabolism
Protein Biosynthesis
Research Papers
RNA, Messenger - metabolism
SARS-CoV-2
Viral Nonstructural Proteins - chemistry
Viral Nonstructural Proteins - genetics
Viral Nonstructural Proteins - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on β-globin, EMCV IRES, and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nt downstream from the mRNA entrance, indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of the Nsp1 NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.
ISSN:0890-9369
1549-5477
DOI:10.1101/gad.350829.123