Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential

Abstract

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.

Fig. 1. N-terminomic analysis of SARS-CoV-2 infection of A549-Ace2 and Vero E6 cells.

a Experimental design. b Viral RNA levels were determined by qRT-PCR (n = 3 biological replicates). c Protein levels were determined based on the TMTpro fractional intensity of the total protein intensity for the unenriched proteomic samples (n = 3 biological replicates). d Infectious virus production (PFU, n = 3 biological replicates). Error bars for b–d represent standard deviation. e A549-Ace2 and f Vero E6 neo-N-terminomic analysis reveals significant increases in peptides corresponding to viral and cellular neo-N-termini, where neo-N-termini must begin from amino acid 2 or later. P values were obtained by two-tailed unpaired t-test, correction for multiple-hypothesis testing to obtain Q-values was performed as described Storey (2002). bRP basic reverse phase fractionation, PFU plaque-forming units. The vertical and horizontal lines correspond to fold change (0.5) cut-off and Q-value (0.05) cut-offs, respectively.

Fig. 2. Proteolysis of viral proteins during SARS-CoV-2 infection.

a Schematic of the SARS-CoV-2 genome and proteome, with the nsp3 (PLP) and nsp5 (MPro) highlighted. Proteolytic processing of SARS-CoV-2 proteins during infection of A549-Ace2 and Vero E6 cells includes b extensive cleavage of the nucleocapsid protein, c N-terminal processing of the ORF3a putative viroporin, and d a novel cleavage site between Y636 and S637 in spike, N-terminal of the FURIN cleavage site. e The abundance of the S637 spike neo-N-terminus increases over the infection timecourse (n = 3, error bars show standard deviation from 3 biological replicates). f This cleavage site is present on a flexible region, C-terminal of the RBD (PDB: 6X6P).

Fig. 3. Several viral neo-N-termini show sensitivity to specific protease inhibitors and a spike 637-proximal mutation alters viral entry in TMPRSS2-ve cells.

a Experimental design for N-terminomics of SARS-CoV-2 infection in the presence of protease inhibitors. b Abundance of viral neo-N-termini in infected cells ± inhibitors. Data normalised to total levels of the relevant viral protein. n = 3 biologically independent samples. Pseudovirus entry assay conducted in c HEK-Ace2 and d HEK-Ace2-TMPRSS2 cells. The infectivity of lentivectors (LV) pseudotyped with the different spike mutants was normalised to that of WT in the HEK-Ace2 cell line. n = 6 (RatG13 n = 5) biologically independent samples. e Western blotting of the pseudovirus stocks used in c and d confirms spike expression and incorporation into lentiviral particles. f Densitometry analysis of spike western blotting data, examining the ratio between uncleaved (S0) and cleaved (S1) portions of the spike protein present in purified pseudotyped lentivirus stocks (n ≥ 3 biological replicates). Boxplot minima/maxima represent the furthest non-outlier datapoints, centre the median, and bounds of box the interquartile range. Outliers are defined as datapoints >1.5 times the interquartile range from the bottom or top of the box. Unpaired Welch’s t-tests, which do not assume equal variance were used for statistical analyses.

Fig. 4. Increased abundance of novel cellular neo-N-termini consistent with SARS-CoV-2 protease consensus sequences suggests viral protease activity on cellular substrates.

Panels a and b show motif analysis highlighting enriched amino acids proximal to the N-terminus of neo-N-termini enriched in SARS-CoV-2-infected A549-Ace2 and Vero E6 cells, respectively. DAU differential amino acid usage. The P5 to P5’ positions for the cleavage sites are shown following the nomenclature of Schechter and Berger. c Consensus motifs for Mpro and PLP. Panels d and e show the relative abundance of cellular neo-N-termini identified as significantly upregulated (n = 3 biological replicates, unpaired two sample t-test, multiple-hypothesis corrected q < 0.05) and matching or resembling the Mpro or PLP consensus motifs from A549-Ace2 or Vero E6 cells, respectively. Sequence match to the consensus is indicated by the pink or green coloring of the P4 to P1 positions of the relevant cleavage sites indicating match to the Mpro or PLP P4, P2, or P1 positions, respectively. f In vitro validation of GFP-tagged PNN, PAICS, and SRC cleavage by SARS-CoV-2 Mpro and PLP, following incubation with 10 M of the respective protease (n = 3). g Cell-based validation of Mpro cleavage of GOLGA3 and PAICS following transfection of SARS-CoV-2 Nsp4-5 plasmid. Tubulin is included as a loading control (n = 3).

Fig. 5. siRNA depletion of potential MPro and PLP substrates results in significant reductions to viral RNA copies and titres in A549-Ace2 cells.

a Experimental design, b Viral RNA copies (n = 3 (control), 5 (NUP107, XRCC1), and 6 (all other samples) biologically independent samples), and c titres in supernatant at 72 h post-infection (n = 6 biologically independent samples), following infection 24 h post-transfection with the indicated siRNA). Boxplot minima/maxima represent the furthest non-outlier datapoints, centre the median, and bounds of box the interquartile range. Outliers are defined as datapoints >1.5 times the interquartile range from the bottom or top of the box. Individual datapoints are shown in grey. Significance was calculated by one-way ANOVA. Blue bars indicate samples with significantly reduced viral RNA copies or titres (p ≤ 0.01). Those not meeting this threshold are shown in red. The control siRNA-treated sample is indicated with a black bar. The limit of detection in the plaque assay was calculated to be 40 PFU/mL (dotted line).

Fig. 6. Inhibitors targeting viral protease substrates reduce SARS-CoV-2 titres in A549-Ace2 cells.

Sorafenib is a tyrosine kinase inhibitor previously shown to inhibit SARS-CoV-2 replication. Bafetinib is a dual ABL/LYN inhibitor with off-target activity against SRC kinase. ML-7 and MLCK target myosin light chain kinase (MYLK/MLCK). Black circles and error bars represent mean and standard deviation. n = 3 biologically independent samples. Red circles indicate individual datapoints. The limit of detection in the plaque assay was calculated to be 40 PFU/mL (dotted line). PFU plaque-forming units.