SARS-CoV-2 3CLpro Main Protease's Role In Cytoskeletal Dynamics And Stealth Intercellular Infection
Nikhil Prasad Fact checked by:Thailand Medical News Feb 22, 2024 9 months, 23 hours, 44 minutes ago
COVID-19 News: The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has prompted extensive research to uncover the intricacies of viral infection and transmission. In a collaborative effort, researchers from the University of British Columbia, National Microbiology Laboratory-Canada, University of Saskatchewan, McMaster University, and the University of Alberta delved into the role of the SARS-CoV-2 3CLpro main protease in driving cytoskeletal reorganization and facilitating stealth intercellular infection. The study findings are covered in this
COVID-19 News report.
Model of 3CLpro-mediated induction of TNTs.
The study results suggest that the enhanced formation of TNTs in SARS-CoV-2 infection is orchestrated by the main protease 3CLpro. SARS-CoV-2 enters ACE2-positive cells via ACE2 with priming by TMPRSS. Once translated, 3CLpro 2 processes host-cell proteins. Cleavage of multiple substrates (validated in this study, shown in green) in the epithelial adherens junction disrupts protein complexes to facilitate cytoskeletal reorganization. Many of these 3CLpro substrates are involved in regulating the Arp2/3 complex: Activation of actin branching causes membrane deformation and protrusion at the plasma membrane to promote TNT formation. In addition, multiple cleavages in the adherens junction activate the zonular signalosome, releasing transcription factors such as YAP1 that translocate to the nucleus and activate expression of genes. This includes substrate genes to compensate for cleaved proteins and re-establish the adherens junction. Cleavage of YAP removes the TEAD binding site, which could allow it to bind in alternative transcription complexes or to antagonize formation of the TEAD transcriptional complex. TRIM28 is a corepressor that acts through KRAB-ZFPs. Cleavage is predicted to relieve repression and result in increased ACE2 expression. Cleavage of proteins in centrosomal complexes may facilitate recruitment to the adherens junction to coordinate microtubule assembly for transport of proteins, cellular components and virus particles for TNT formation and immune-protected infection of other cells.
Identification of Interactors and Substrates
To understand the molecular mechanisms underlying SARS-CoV-2 infection and dissemination, the researchers employed Inactive Catalytic Domain Capture (ICDC) and proteomics. They identified 259 interactors of the 3CLpro main protease, with 145 associated with the cytoskeleton and its organization. Enzyme kinetic specificity constants were determined for 139 3CLpro cut-sites in 43 interactors, leading to the validation of 29 efficiently-cleaved substrates in vitro, in SARS-CoV-2-infected human lung cells, and in post-mortem COVID-19 lungs.
Cytoskeletal Remodeling and Tunnelling Nanotube Formation
The researchers observed that 3CLpro cleavage of adherens junction proteins initiated cytoskeletal rearrangement,
activating zonular signaling. This process removed subcellular localization motifs, triggering the translocation of nuclear proteins TRIM28 and NUMA1 to adherens junctions, as well as YAP1 in the Hippo pathway to the nucleus. These cytoskeletal remodeling events rapidly generated tunnelling nanotubes (TNTs) connecting lung epithelial cells, facilitating stealthy viral transmission to distant cells.
Viral Entry and Tunnelling Nanotube Formation
SARS-CoV-2 enters cells by exploiting host proteases such as TMPRSS2, cathepsins B and L, and furin to prime the viral spike protein. Tunnelling nanotubes (TNTs), tubular actin-rich protrusions, have been observed in infected lung epithelial cells. The study indicates that 3CLpro activity triggers TNT formation in infected cells, establishing intercellular immune-privileged conduits for viral transmission. This mechanism may impede the effectiveness of immune recognition and vaccines aimed at reducing viral transmission.
Interactome Analysis and Cytoskeletal Proteins
The study extensively analyzed the 3CLpro interactome, revealing that cytoskeletal and RNA-binding proteins dominate the interactions. Of the 259 interactors, 56% were associated with cytoskeletal organization and/or cell protrusions. Further analyses indicated that numerous proteins in the interactome regulate Arp2/3 activity, impacting actin branching and providing the force for plasma membrane deformation and TNT initiation.
3CLpro Substrates and TNT Formation
The researchers identified 126 3CLpro-preferred cleavage motifs in 259 interactors, with 59 efficiently-cleaved cut sites. Among these, non-muscle myosin II (NMII) was identified as a key player in TNT formation. Efficient cleavage of myosin-9 by 3CLpro destabilized the cytoskeleton and initiated TNT formation. The study also demonstrated that TNT formation was inhibited by the addition of the 3CLpro inhibitor GC376, further emphasizing the role of 3CLpro in this process.
Adherens Junction Disruption and Zonular Signalosome Activation
The study uncovered the extensive role of 3CLpro in disrupting adherens junction proteins, including cleavage of zyxin, LIMA1, LASP1, and nebulin. Cleavage of these proteins contributed to the reorganization of actin filaments for TNT formation. Additionally, 3CLpro cleavage activated the zonular signalosome, leading to changes in the subcellular localization of critical signaling proteins, further facilitating viral spread.
Therapeutic Implications
While current vaccines primarily target the SARS-CoV-2 spike protein, the study emphasizes the need for new therapeutic targets to reduce viral transmission. The researchers highlight the critical role of the 3CLpro main protease in SARS-CoV-2 infection, extending beyond viral polyprotein processing to include cleavages of host cell proteins. Inhibitors such as Nirmatrelvir (PAXLOVIDTM) and GC376 have demonstrated efficacy, underscoring the importance of 3CLpro in the viral life cycle.
Conclusion
This comprehensive study sheds light on the multifaceted role of the SARS-CoV-2 3CLpro main protease in driving cytoskeletal reorganization and TNT formation for stealthy intercellular infection. The findings provide valuable insights into potential therapeutic targets for mitigating viral transmission. Understanding the intricate dance between the virus and host cell machinery is crucial for developing effective strategies to combat COVID-19 and future coronaviruses.
The study findings were published on a preprint server and are currently being peer reviewed.
https://www.researchsquare.com/article/rs-3918469/v1
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