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.

Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome

“…However, no further biochemical or physiological validation was performed. In addition, Meyer et al (2021) very recently reported cleavage of NUP107 (Table 1) at position Gln 35 in SARS-CoV-2-infected A549-ACE2 cells and GOLGA3 at Gln 365 (Table S1), and ATAD2 at Gln 949 (Table 1), which we also found. In their study, GOLGA3 cleavage was elegantly validated in 3CL pro transfected cells, whereas NUP107 and ATAD2 cleavages were attributed to 3CL pro based on the cleavage logo but without direct evidence.…”

“…We also demonstrate similarities and divergence at P2 from the dominant leucine specificity (Figure 1C) previously reported in the SARS-CoV-2 polyprotein (Scott et al, 2021), peptides (Rut et al, 2021), and monkey and human proteins (Koudelka et al, 2021;Meyer et al, 2021). In the polyprotein, P2-Val and P2-Phe each occur once.…”

“…Many large-scale analyses of the SARS-CoV-2 infected-cell transcriptome (Stukalov et al, 2021), proteome (Stukalov et al, 2021), phosphoproteome (Bouhaddou et al, 2020) and interactomes (Gordon et al, 2020;Stukalov et al, 2021) are described. With only 14 substrates reported in SARS-CoV-2 infection (Meyer et al, 2021;Moustaqil et al, 2021), the 3CL pro human substrate repertoire, also known as the degradome (Lo ´pez-Otı ´n and Overall, 2002), is not well understood. Thus, the opaque contribution of 3CL pro to overwhelming the host cell machinery remains understudied.…”

SARS-CoV-2 PLpro whole human proteome cleavage prediction and enrichment/depletion analysis

“…FOXP3 was also the only experimentally validated protein to be successfully predicted here. [18][19][20] This indicates that, as with 3CLpro prediction, any inaccuracies in prediction methods are amplified when applied to the entire human and must be experimentally verified, however enrichment analyses are robust against this variability. [1]…”

Drug Combinations as a First Line of Defense against Coronaviruses and Other Emerging Viruses

“…Hence, some endosome-targeting drugs may also act directly on viral glycoproteins ( 69 , 176 – 179 ), i.e., be both HTAs and DAAs. Other host factors to consider for targeting include host proteases that prime viral glycoproteins for fusion ( 37 , 38 , 119 , 120 ), other host proteins involved in virus entry ( 180 ), host cell kinases ( 181 – 184 ), and host proteins involved in viral RNA production ( 185 , 186 ), nuclear export ( 187 , 188 ), and virus assembly and egress ( 189 , 190 ). Given common viral infection strategies, the possibility exists for cross-family drug cocktails.…”