The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation

Abstract

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.

Fig 1. Proximal Interactome of 17 SARS-CoV-2 proteins.

A) Schematic of BioID workflow. B) Curated network of SARS-CoV2 virus-host protein associations (SAINT ≥ 0.9) obtained from BASU BioID. Coronavirus proteins are labeled in light blue and virus-host interactions are connected by red edges, while host-host protein interactions obtained from high confidence STRING interactions are labeled in grey. Highlighted node clusters of similar function, including 26S proteasome components (black), MHC Class I (red), nuclear pore (dark blue), RNA-binding (maroon), SNARE complex (purple), translation initiation complex (green) proteins were selected based on GO term analysis. C) Selected biological process GO term enrichment; enrichment scores are given as -Log10 p-values. Selected GO terms are nuclear pore organization, translational initiation, endosomal transport, and RNA splicing. D) Heatmap of molecular function GO term enrichment of SARS-CoV-2 proteins. All presented GO terms have a -Log10 p-value >3 for the Nucleoprotein, the listed non-structural proteins, or the listed open reading frames or a -Log10 p-Value >5 for the M membrane protein.

Fig 2. Localization of SARS-CoV-2 proteins.

A) Heatmap of cell component GO term enrichment of SARS-CoV-2 proteins. B) Western blots of SARS-CoV-2 viral protein-expressing HEK293T cell fractions; whole cell lysate (WCL), cytosol, cytosol/membrane, nucleus/membrane and nucleus fractions. Alpha-tubulin, calnexin, and histone H3 were used as fractionation controls for cytosol, membrane, and nucleus respectively. Schematic C) and table D) depicting the predicted location of all SARS-CoV-2 proteins surveyed in this study based on both the BioID and fractionation analysis.

Fig 3. Immunofluorescent staining of SARS-CoV-2 proteins in infected cells.

A549 cells expressing ACE2 were infected for 48 hours with SARS-CoV-2 WA1 strain, fixed, and stained with the indicated viral (green) and host proteins (red) as well as nuclear staining by Hoescht (blue). A) NSP1, B) ORF6, C) NSP14.

Fig 4. COVID disease risk eGenes proximal to viral proteins.

A) Table of GWAS risk SNPs which also scored as BioID hit. B) Map of connectedness of eGenes (Mauve) with BioID interactors (Gray) and the corresponding viral proteins (Purple). eGenes also identified by BioID are outlined in black. GWAS-identified eGenes-associated with antiviral response, cell-cycle, transcription, and translation are also highlighted. C) Virtual 4-C plot showing chromatin contact between TRIM4 promoter and linked COVID disease risk SNP (rs1569055). Genome tracks showing ATAC peaks and contact loops of GM12878, Naïve T, Th-17, and T-reg cells.

Fig 5. NSP1 and ORF6 disruption of host translation and innate immune signaling.

A) Curated map of NSP1 proximal interactors. Highlighted are host proteins involved in translation initiation. B) Effect of NSP1 on translation of in vitro transcribed, capped polyadenylated transcripts containing 5’ UTRs from SARS CoV-2, IFIT1, and ISG15 as well as IRES elements from XIAP1, APAF1, and CRPV. Data shown is the average of three independent experiments and significance was calculated using Student’s T Test where * indicates p value<0.005. Curated maps of ORF6 C) and ORF9b D) proximal proteins showing nuclear pore protein complex association with ORF6 and MAVS association with both ORF6 and ORF9b. E) Effect of ORF6 and ORF9b on IFNB1 promoter activity after RIG-I 2-CARD induction. Normalized luciferase shown is the ratio of nano luciferase/firefly luciferase normalized to empty vector control. Data shown is the average of three independent experiments.

Fig 6. BioID and SILAC MS identify candidate targets for the viral protease NSP5.

A) Comparison of two biological replicates of protein abundance in HEK293T cells expressing either NSP5 wild type (WT) or the catalytically inactive NSP5^C145A mutant by log₂ fold change. B) Map of NSP5 proximal interactome (Gray) overlaid with host proteins decreased in abundance in SILAC (Red). CDNK2AIP was detected as both a BioID hit and decreased in abundance in SILAC. C) Peptide cleavage assay of four sequences from SARS-CoV-2 polyprotein PP1AB and the indicated host genes: CWC22, CDNK2AIP, FANCD2, P53. Normalized FRET signal is shown comparing HEK293T cells expressing either wild type NSP5 or NSP5^C145A. PP1ab and CWC22 mutant (mut) sequences contain nucleic acid changes that result in QS→AS mutation in the peptide sequence. Data shown is representative of three independent experiments and significance was calculated using Student’s T Test. * Indicates p value <0.05, NS not significant. Dose-dependent effect of coronavirus protease inhibitor G376 on cleavage of peptide encoded by PP1ab-2 D) and FANCD2-2 E) sequences.

Fig 7. SARS-CoV-2 proximal proteins in translation and interferon activation.

Model of SARS-CoV-2 antagonism of host antiviral response. ORF6 protein inhibits RLR signaling leading to decreased type I interferon and ISG transcription, M protein through TRIM4 interactions may also alter host response. NSP1 disrupts host translation of transcripts containing both ISG 5’ UTR and stress responsive IRES elements.