Proximity-dependent biotinylation detects associations between SARS coronavirus nonstructural protein 1 and stress granule–associated proteins

Yevgeniy A Gerassimovich,Samantha J Miladinovski-Bangall,Kaitlin M Bridges,Linkel Boateng,Lauren E Ball,Homayoun Valafar,Anita Nag

doi:10.1016/j.jbc.2021.101399

Abstract

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host mRNAs. To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)–associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras GTPase-activating protein SH3 domain–binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite–induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1.

Highlights

Severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) are enveloped viruses containing a long, single, and positive-stranded RNA genome [1,2,3]
We further report that several pre-mRNA processing proteins and stress granule (SG)–associated factors were identified in addition to proteins involved in mRNA translation
To capture proteins that dynamically interact with nsp1 in the native environment of the cell and the cellular pathways that facilitate the function of nsp1s, we performed proximity labeling using an engineered biotin ligase (BioID2) fused to nsp1 in human embryonic kidney 293 (HEK293) cells (Fig. S1)

Summary

Introduction

Severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) are enveloped viruses containing a long, single, and positive-stranded RNA genome [1,2,3]. Mapping of only the C-terminal region of nsp into the cryo-electron microscopy structure of the nsp1-bound 40S complex determined that the structurally undefined C-terminal domain of the protein folds into a defined helix–turn–helix structure to bind the mRNA binding site of the 40S ribosome [9, 10] In this experiment, Schubert et al [9] studied thermodynamically stable complexes between bacterially expressed nsp and human embryonic kidney 293 (HEK293) cell lysate. Genome-wide interactions of coronavirus proteins have been identified using MS in cells expressing SARS-CoV-2 proteins [18], in-cell stable isotope labeling using subgenomic SARS-CoV, and using biotin labeling of mouse hepatitis virus–infected cells [19,20,21] While these studies provided valuable information, none of them focused exclusively on nsp and compared the binding of nsp of SARS-CoV and SARS-CoV-2 to host proteins. We further report that several pre-mRNA processing proteins and stress granule (SG)–associated factors were identified in addition to proteins involved in mRNA translation

Methods

Results

Conclusion