Investigation of the role of cyclins and cyclin-dependent kinases in host restriction of SARS-CoV-2 infection

In a recent study posted on bioRxiv* Preprint server, researchers investigated changes in levels of cyclins and cyclin-dependent kinases (CDKs) in host cells caused by coronavirus disease 2019 (COVID-19).

Both cyclins and CDKs are important regulators of the host cell cycle.lots of coronavirus (CoVs) contain Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which migrates and degrades cyclins in host cells for efficient replication.

A recent study investigating the phosphorylated proteome profile of SARS-CoV-2 infected VEROE6 cells revealed that SARS-CoV-2 reduced the activity of CDK1 and CDK2. In this way, the virus arrests the host cell cycle during the deoxyribonucleic acid (DNA) synthesis (S) and cell proliferation (G2) phases.

It is noteworthy that pathogenic viruses use nucleotides synthesized in host cells for replication. It also increases the DNA repair processes and replication proteins that are essential for viral replication by blocking the arrest of the host cell cycle.

About research

In this study, researchers used VERO and A549 alveolar type II (AT2) cells to collect extensive data on changes in the SARS-CoV-2 cell cycle.

They used immunofluorescence to analyze the cell distribution of cyclins D1, D3, and A2 in infected VERO AT2 cells. Fluorescence intensity reflected the cyclin nucleus and cytoplasmic (N / C) ratios of uninfected and infected cells. In addition, researchers measured D-cyclin levels in infected cells in the absence or presence of the protease inhibitors carbobenzoxy-Leu-Leu-leucinal (MG-132) and bortezomib.

Small interfering RNA (siRNA) reduces cyclin D and A2 levels by more than 80% 48 hours after transfection of A549 AT2 cells compared to non-targeted controls. Cyclin-depleted cells were exposed multiple times to delta, alpha, and wild-type SARS-CoV-2 mutants to investigate the functional role of D-cyclin in the pathogenesis of SARS-CoV-2.

The team investigated the phenotype of cell cycle arrest using the fluorescent ubiquitinated cell cycle indicator (FUCCI) (or sensor) of VERO and A549 AT2 cells. Finally, they used flow cytometry to visualize the G1, early S, S, G2, and mitotic (M) stages.

Investigation result

Research results show that SARS-CoV-2 infection caused the translocation of cyclin D1 and cyclin D3 from the host cell nucleus to the cytoplasm for proteasome degradation, restoring their assembly and release of newly generated virions. rice field.

Expression of the Fucci sensor helped researchers quantify the N / C ratio of D-cyclin staining at various cell cycle stages. The results showed that cyclin D3 and cyclin D1 in SARS-CoV-2 infected cells relocalized from the nucleus to the cytoplasm for degradation, regardless of the stage of the cell cycle. Interestingly, SARS-CoV-2 only induced degradation of the cyclin D protein. Therefore, analysis of other cyclins in VERO AT2 cells showed no changes in cyclins A, B, or E.

Cyclin A promotes transition to phase S and mitosis, cyclin B accumulates during cell proliferation (G2), and cyclin E prevents transition to phase S. In addition, the specific cell cycle stages in which cells stop appear to be cell type-dependent.

When cyclin D1 and cyclin D3 levels decrease, the host cell cycle stops in G1 phase instead of S / G2 phase. In the absence of cyclin D, A549 AT2 cells arrest early in S phase and VERO AT2 cells arrest in S / G2 or M phase, indicating the role of other factors in cell cycle arrest during COVID-19.

In particular, the cyclin D protein is constantly relocalized from the nucleus to the cytoplasm and degraded at all cell cycle stages in COVID-19. Conversely, CDK expression levels did not change. The authors suspected that the phosphorylation state of the CDK may have influenced the progression of the cell cycle. However, this study has not investigated this effect.

SARS-CoV-2 requires M, E, and nucleocapsid (N) proteins for virion assembly in the endoplasmic reticulum (ER) -Golgi intermediate compartment system. The authors noted the role of cyclin D3 in the spread of SARS-CoV-2 and defects in viral assembly. More importantly, it has emerged as a new interacting agent with SARS-CoV-2 envelope (E) and membrane (M) proteins.

Cyclin D3 binds to the SARS-CoV-2M protein and inhibits SARS-CoV-2 assembly and diffusion. Conversely, host cells carry the SARS-CoV-2 spike (S) protein in the presence of M and E. However, the ability to translocate to the membrane and form syncytium is diminished due to cyclin D3. The data support the hypothesis that cyclin D3 is a limiting factor that interferes with the role of M and E in the SARS-CoV-2 assembly.

Conclusion

This study highlights how the cyclin D protein interferes with the SARS-CoV-2 E and M proteins, impairing efficient virus assembly and replication. Therefore, SARS-CoV-2 has evolved a strategy to degrade cyclin D3 for more efficient virion assembly and replication. This significant phenomenon requires extensive investigation across all SARS-CoV-2 mutants in order to develop a universal antiviral target for SARS-CoV-2.

*Important Notices

bioRxiv publishes unpeer-reviewed preliminary scientific reports and should not be considered definitive, guide clinical / health-related behaviors, or be treated as established information.