A new study led by researchers from the SLAC National Accelerator Laboratory at Stanford University and the National Virtual Biotechnology Laboratory at the US Department of Energy-Washington has found the SARS-CoV-2 main protease Mpro also called 3CLpro (3-chymotrypsin like protease ) is able to cleave a key human immune signaling protein called NEMO (NF-κB Essential Modulator) in the process severing certain critical immune pathways and disarming certain immune responses.
The study findings were published in the peer reviewed journal: Nature Communications. https://www.nature.com/articles/s41467-022-32922-9
As of April 2022, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has caused over 507 million confirmed cases of COVID-19, more than 6 million deaths (covid19.who.int/), and the global economy to contract by 3.5% in 20201.
Unlike previous betacoronavirus outbreaks, SARS-CoV-2 has spread to every country, which has provided the urgency and impetus to develop and rapidly distribute therapeutics to reduce the spread of the virus, including RNA-based vaccines.
However, many societal impediments, the emergence of variants with enhanced fitness and breakthrough infections have prevented herd immunity from being reached from vaccination coverage, and prove that SARS-CoV-2 eradication is challenging. Therefore, more broadly protective vaccines and effective therapeutic approaches will need to be implemented to steadily reduce the risk of severe illness and prevent future zoonotic outbreaks.
Current strategies to inhibit viral transmission and to reduce the severity of the disease include disrupting the lifecycle of the pathogen2,3. SARS-CoV-2 is an enveloped betacoronavirus with a single stranded, positive-sense, 29 kb RNA genome that encodes several open reading frames. ORF1a and ORF1b encode the polyproteins that are processed to generate the 15 nonstructural proteins of SARS-CoV-2.
These include the papain-like protease (PLpro) and the 3C-like protease (3CLpro), which are required to execute the viral life cycle and inhibit the host immune response4,5. These proteases therefore represent high-value targets for treatment of COVID19, which is supported by the emergency use authorization granted by the Food and Drug Administration for PaxlovidTM that includes nirmatrelvir, a 3CLpro inhibitor component of the drug.
The Protein Data Bank (RCSB PDB) is replete with structures of wild-type (WT) SARS-CoV-2 3CLpro6,7. 3CLpro WT homodimers are a 67.60 kDa, heart-shaped complex6,8,9,10. Each 3CLpro chain consists of three domains. Domain I (aa. 8–101) and Domain II (aa. 102-184) have a predominantly β-sheet structure, form the active site, and contribute to dimerization. Domain III (aa. 201-303) is substantially α-helical and is the primary determinant of dimerization 8,9,11.
The active site of WT 3CLpro contains the catalytic dyad of His41 and Cys145. Prior to substrate-binding, the 3CLpro active site is primed with protonated His41 and a thiolate anion on Cys145. After substrate-binding, the thiolate anion prosecutes nucleophilic attack at the main chain carbonyl carbon of the P1 residue (immediately preceding the substrate scissile bond)12,13 This leads to heterolytic fission of the scissile bond, followed by active site regeneration.
Functionally, 3CLpro recognizes a hydrophobic substrate residue at P2 (usually Phe or Leu), a Gln at P1, and Ser, Val, Asn, or Ala residues at P1’. This recognition motif is found in multiple sites of the viral polyproteins, which are cleaved by 3CLpro to form mature nsp5-16. This consensus sequence is also present in proteins of the host innate immune pathway and therefore 3CLpro may blunt the immediate antiviral immune response via proteolysis 14.
The NF-κB essential modulator (NEMO)15 is one of the immune proteins that can be cleaved by 3CLpro14. NEMO has been shown to be cleaved by 3CLpro from feline infectious peritonitis virus and porcine epidemic diarrhea virus 16,17. Recently, N-terminomics experiments also confirmed, in vitro, the cleavage of NEMO by SARS-CoV-2 3CLpro14.
NEMO is necessary for activating NF-κB during the canonical NF-κB response signaling pathway, which is a critical first response to viral infection. A disrupted NF-κB pathway is a hallmark of chronic inflammatory diseases 18, which suggests that NEMO cleavage by 3CLpro and the downstream dysregulation of NF-κB could contribute to the enhanced inflammatory response observed in COVID-19 patients15.
Remarkably, consistent with in vitro and in vivo data, Wenzel et al. recently proposed that cleavage of host cell NEMO by 3CLpro is connected to microvascular pathology observed in the brains of COVID-19 patients19. Therefore, understanding the molecular basis of NEMO inactivation by 3CLpro can be a platform to develop therapeutic strategies for alleviating symptoms of COVID-19 including pathology in the central nervous system.
The structure of 3CLpro in complex with a NEMO-derived heptapeptide substrate was previously solved for the Porcine Epidemic Diarrhea Virus, an alphacoronavirus20. Molecular docking and comparative structural analyses suggest that NEMO similarly binds to SARS-CoV-2 3CLpro in a pose that favors proteolysis 21.
However, even a few differences in 3CLpro residues, for example, between SARS-CoV and SARS-CoV-2 3CLpro, were shown to significantly change substrate preferences14, enforcing the importance of obtaining a high resolution structure of SARS-CoV-2 3CLpro bound to human NEMO and identifying non-conserved interactions.
In this work, we first showed that WT SARS-CoV-2 3CLpro can cleave the 33-residue peptide substrate, NEMO215–247 using in vitro assays. To explore the molecular basis of this interaction, we solved a 2.50 Å crystal structure of a SARS-CoV-2 3CLpro active site cysteine variant, C145S, in complex with the human decapeptide substrate NEMO226–235.
This represents the first structure of SARS-CoV-2 3CLpro bound to a human substrate protein. Using this structure as a starting point, extensive molecular dynamics simulations, quantum mechanics calculations, and machine learning-based predictions indicated that the few differences in NEMO and 3CLpro across host species and human-infecting betacoronaviruses significantly change the stability of the complex formed between these proteins. Finally, we discuss how ablation of NEMO via proteolysis connects with COVID-19 as a systemic disease.