Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit.

Highlights: · ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species· Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways· Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function· Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection.

Figure 1:. ChIRP-MS identifies host and viral proteins associated with the SARS-CoV-2 RNA genome in infected cells.

(A) Schematic of the ChIRP-MS protocol. (B) SDS-PAGE analysis of total protein samples enriched using SARS-CoV-2 targeting biotinylated oligonucleotides from Mock (uninfected) cells or cells infected for 24 or 48 hours with SARS-CoV-2 in both Huh7.5 (top) and VeroE6 (bottom) cells. (C)Quantification of the percentage of reads mapping to SARS-CoV-2 gRNA (ORF1a/b) versus the subgenomic RNA (sgRNA) before and after pulldown. (D)RNA-seq coverage of the SARS-CoV-2 genome before and after pulldown. (E)Structure of the SARS-CoV-2 genome. (F) Enrichment of each viral protein in Huh7.5 and VeroE6 cells at both time points.

Figure 2:. Changes in the SARS-CoV-2 associated proteome across time points and species.

(A) Significantly enriched proteins in Vero cells after viral RNA pulldown at 24 and 48 h.p.i. (B)Significantly enriched proteins in Huh7.5 cells after viral RNA pulldown at 24 and 48 h.p.i. (C) Conservation of enriched proteins between time points (left, middle) and species (right). (D) Cytoscape network representation of the SARS-CoV-2 associated human proteome. Colors indicate ChIRP enrichment in Huh7.5 cells 48 h.p.i.

Figure 3:. Comparison of the SARS-CoV-2 associated proteome to that of other RNA viruses.

(A) Principal component analysis of ChIRP enrichments in human cells across time points and viruses. (B) Upset plot comparing expanded interactomes of SARS-CoV-2, ZIKV, DENV, and RV in human cells at 48 h.p.i. (C) Top: Cellular Components GO terms enriched in the expanded interactome of each virus. Bottom: Binding Protein GO terms enriched in the expanded interactome of each virus. (D)Comparison of proteasome subunits and proteasome accessory factor associations across viruses.

Figure 4:. Cellular context of expanded interactomes across viruses.

Selected groups of proteins, their enrichment in SARS-CoV-2, Zika, Dengue, and Rhinovirus ChIRP, and their approximate subcellular localization. Heat map colors indicate the log₂ ChIRP-MS enrichment values. Each heatmap has a separate scale bar.

Figure 5:. Integration of ChIRP-MS and functional genomic data suggest novel pro- and anti-viral host factors.

(A) High confidence SARS-CoV-2 interactome overlaid on VeroE6 CRISPR screen data. (B) Expanded SARS-CoV-2 interactome overlaid on CRISPR screen data. (C)Comparison of CRISPR guide RNA (sgRNA) residuals for significant hits (fdr <= 0.05) of all genes (left, black), genes present in the high-confidence SARS-CoV-2 RNA interactome (purple, middle), or genes present in the expanded SARS-CoV-2 RNA interactome (right, blue). P values computed from Mann-Whitney test. (D) High confidence SARS-CoV-2 human interactome network colored by enrichment or depletion in CRISPR screen. (E) sgRNA residuals for CRISPR hits identified in (B) grouped by cellular pathways in Figure 4. Individual CRISPR guides are represented by black lines. The average of these is shown in red. (F) Inter-virus comparison of shared ChIRP-MS / CRISPR hits identified in (A). (G) sgRNA residuals for top 20 sensitizing hits (left) and for all significant resistance hits (right) identified in (B).

Figure 6:. SARS-CoV-2 associated proteins and RNAs nominate the mitochondria in viral pathogenesis.

(A) Enriched host RNAs after vRNA pulldown in VeroE6 cell line 48 h.p.i. (B)Enriched host RNAs after vRNA pulldown in Huh7.5 cell line 48 h.p.i. (C) Electron microscopy (EM) of HBEC cells uninfected (left, Mock) or infected by SARS-CoV-2 (right). Selected mitochondria indicated with arrowheads in the inset. (D) Quantification of mitochondria size by EM in five ciliated (infected) cells. (E) Mitochondrial proteins which are present in the expanded interactome of at least one virus and their conservation across viruses. Segments of the circle, from smallest to largest, correspond to proteins encoded by the mitochondrial genome, components of the mitochondrial ribosome, and proteins encoded by the nuclear genome which are localized or associated with the mitochondria. Enrichment (log₂ FC) scale is capped at 2. Proteins which are significant hits in the CRISPR screen data in Figure 5 are indicated with red labels. (F)CRISPR sgRNA residuals for mitochondrially annotated CRISPR hits labeled in red in E.