Characterization and functional interrogation of SARS-CoV-2 RNA interactome

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 pandemic, which has caused a devastating global health crisis. The emergence of highly transmissible novel viral strains that escape neutralizing responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2 biology and to develop additional therapeutic strategies. Using a comprehensive identification of RNA binding proteins (RBP) by mass spectrometry (ChIRP-M/S) approach, we identified 142 high-confidence cellular factors that bind the SARS-CoV-2 viral genome during infection. By systematically knocking down their expression in a human lung epithelial cell line, we found that the majority of the RBPs identified in our study are proviral factors that regulate SARS-CoV-2 genome replication. We showed that some of these proteins represented drug targets of interest for inhibiting SARS-CoV-2 infection. In conclusion, this study provides a comprehensive view of the SARS-CoV-2 RNA interactome during infection and highlights candidates for host-centered antiviral therapies.

Uncovering viral RNA–host cell interactions on a proteome-wide scale

“…Therefore, it is possible that DNA-PK components also contribute to vRNA metabolism. In agreement, XRCC5 depletion attenuates SARS-CoV-2 infection, hinting at a proviral role for this DNA-PK component [45]. In summary, the exact roles of DNA-PK and, more broadly, DDR in the life cycle of RNA viruses awaits better characterisation.…”

“…Abbreviations: 4SU, 4-thiouridine; ActD, actinomycin D; ChIRP-MS, comprehensive identification of RNA-binding proteins by mass spectrometry; CLAMP, crosslinkassisted messenger ribonucleoprotein purification; Fvo, flavopiridol; HyPR-MS, hybridization purification of RNAprotein complexes followed by mass spectrometry; mRNP, messenger ribonucleoprotein; RAP-MS, RNA antisense purification and quantitative mass spectrometry; SPRI, solid-phase reversible immobilisation; TUX-MS, thiouracil cross-linking mass spectrometry; VIR-CLASP, viral cross-linking and solid-phase purification; vRIC, viral RNA interactome capture. See also [25][26][27][40][41][42][43][44][45][46][50][51][52][53].…”

“…Isolation of protein-vRNA complexes After 'freezing' protein-RNA interactions, vRNA and its covalently linked proteins are isolated. A common strategy to achieve this is to capture the vRNA specifically using antisense probes, which can be single probes [hybridization purification of RNA-protein complexes followed by mass spectrometry (HyPR-MS)] [27,40] or multiprobe sets [RNA antisense purification-quantitative MS (RAP-MS)] [41][42][43], comprehensive identification of RNA-binding proteins by MS (ChIRP-MS) [44][45][46]. Although multiprobe sets maximise vRNA recovery due to multiple simultaneous probe-target hybridisation events, each probe will have its own set of off-target interactions that will add to the overall experimental noise (Box 1).…”

Computational Mapping of the Human-SARS-CoV-2 Protein-RNA Interactome

“…Multiple recent studies show that SARS-CoV-2 RNAs extensively interact with both pro-and anti-viral host RBPs during its life cycle [Flynn et al (2021), Schmidt et al (2021), Lee et al (2021), Labeau et al (2021)]. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), Flynn et al [Flynn et al (2021)] identified a total of 229 vRNA-bound host factors in human Huh7.5 cells with prominent roles in protecting the host from virus-induced cell death.…”

SARS-CoV-2 RNA: Exclusive friends and common foes

“…Extensive efforts have been undertaken to characterize SARS-CoV-2 proteins and their interactions with host factors, yet a gap remained in the understanding of how cellular factors interact with the viral genome. Recent studies have endeavored to elucidate the interactions between SARS-CoV-2 viral RNA (vRNA) and host cell proteins (Flynn et al, 2021;Labeau et al, 2021;Lee et al, 2020;Schmidt et al, 2021). In this issue of Cell, Flynn, Belk, et al explore the vRNA interactome during SARS-CoV-2 infection using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) and functionally analyze the identified RNA-host protein interactions (Figure 1) (Flynn et al, 2021).…”