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

Abstract

The main viral protease (3CL pro ) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CL pro by TAILS substrate-targeted N-terminomics. We identify >100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CL pro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CL pro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike healthy lung cells. The 3CL pro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate

“…M pro inhibitors can effectively block SARS-CoV-2 replication in cell culture, demonstrating M pro is a valid drug target (4)(5)(6)(7)(8). SARS-CoV-2 M pro has been shown to preferentially recognize the A-X-L-Q-(A/S) consensus sequence (where X is any amino acid), with cleavage occurring after the glutamine (9,10). Interestingly, other coronaviruses including the related SARS-CoV and MERS-CoV share similar substrate specificity with SARS-CoV-2 M pro , suggesting that inhibitors of SARS-CoV-2 M pro could serve as broad spectrum antiviral drugs against future epidemic or pandemic causing coronaviruses (4,11).…”

Predicted coronavirus Nsp5 protease cleavage sites in the human proteome

“…Many other potential cleavage sites have been identified by Koudelka et al and Pablos et al, where N-terminomics was used to identify possible cleavage sites, after cell lysate was incubated with various coronavirus Nsp5 proteases [ 45 , 54 ]. Out of the 383 unique peptides identified by Koudelka et al where a glutamine was at P1, NetCorona predicted that 167 (44%) of them would be cleaved (Additional file 1 : Table S6).…”

“…Lysine and arginine appear in many cleavage sites predicted by NetCorona, meaning that cleavage by trypsin may mask true cleavage sites by artificially generating a N-terminus proximal to a P1 glutamine residue. Only 38 cleavage sites were commonly identified by both Koudelka et al and Pablos et al using similar N-terminomics approaches, out of the hundreds of potentially cleaved peptides that each study identified [ 45 , 54 ], likely as these studies used different cell lines and thus different proteins will be expressed. Meyer et al point out that the lysate-based method used by Koudelka et al and Pablos et al strips proteins of their subcellular context, which may lead to observed cleavage events that are not possible in vivo during infection [ 46 ].…”

“…Nsp5 also appears to accommodate more diversity at P1’ and P3 than what NetCorona was trained on, based on the naturally occurring SARS-CoV-2 pp1ab variants we report here. That histidine and methionine can be accommodated at P1, and phenylalanine at P2, albeit unfavorably, further adds complexity to what human proteins may be cleaved by Nsp5 [ 17 , 26 , 45 , 54 , 67 ]. The results of this study are instead meant to provide guidance for in vitro experimental design and interpretation of experimental results, in addition to suggesting proteome-wide trends in molecular pathways that Nsp5 from coronaviruses may disrupt to evade the immune system, or augment to favor viral replication through targeted cleavage of human proteins.…”

“…Coronavirus Nsp5 antagonism of IFN is not yet clear [ 32 , 48 – 50 ], but in vitro evidence supports SARS-CoV-2 Nsp5 mediated cleavage of TAB1, NLRP12, RIG-I, and RNF20 which are involved in innate immunity [ 51 – 53 ]. Hundreds of potentially cleaved peptides containing the Nsp5 consensus sequence appeared when lysate from human cells were incubated with recombinant Nsp5 from SARS-CoV, SARS-CoV-2, or hCoV-NL63, indicating a significant potential for Nsp5 mediated disruption of host proteins [ 45 , 54 ]. Similarly, the abundance of potentially cleaved peptides containing a Nsp5 consensus sequence was increased in cells infected in vitro with SARS-CoV-2, which was dependent on the cell type studied [ 46 ].…”