The SARS-CoV-2 RNA\u2013protein interactome in infected human cells

Nora Schmidt,Lars Dölken,Cornelius Schneider,Jörg Vogel,Matthias M Zimmer ,Neva Caliskan,Hasmik Keshishian,Simone Werner,Jochen Bodem,Sebastian Zielinski,Utz Fischer,Luisa Kirschner,Thomas Hennig,Caleb A Lareau,Randy Melanson,Eric S Lander,Jens Ade,Steven A Carr,Sabina Ganskih,Yuanjie Wei,Mathias Munschauer

doi:10.1038/s41564-020-00846-z

Abstract

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.

Highlights

The rapid spread of a new severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) around the globe has led to a worldwide spike in a SARS-like respiratory illness termed coronavirus disease 2019 (COVID-19)[1]
To purify SARS-CoV-2 RNAs and the complement of directly crosslinked cellular proteins from infected human cells, we designed a pool of biotinylated DNA oligonucleotides antisense to the positive-sense SARS-CoV-2 RNA and its subgenomic messenger RNAs
In addition to Huh[7] cells, we evaluated all inhibitors in Calu[3] cells, a human lung epithelial cell line that is naturally susceptible to SARS-CoV-2 infection

Summary

Introduction

The rapid spread of a new severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) around the globe has led to a worldwide spike in a SARS-like respiratory illness termed coronavirus disease 2019 (COVID-19)[1]. SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus that, upon infection of a host cell, deploys a ‘translation-ready’ RNA molecule, which uses the protein synthesis machinery of the host to express a set of viral proteins crucial for replication[2]. Mapping of the interactions between viral and host proteins has revealed cellular pathways relevant to productive infection[12]. These studies cannot reveal how viral RNA is regulated during infection or how host cell RNA metabolism is remodelled to enable virus replication[13]. RNA antisense purification and quantitative mass spectrometry (RAP–MS) combines UV crosslinking with a highly denaturing purification procedure and is ideally suited to capture and identify only those proteins that bind directly to SARS-CoV-2 RNAs14,15

Methods

Results

Conclusion