Researchers have found the host protease CAPN2 to be a novel host factor that aids SARS-CoV-2 infection

In a recent study posted on Bio Rxiv*Preprint server, researchers investigated the potential of the calpain 2 (CAPN2) molecule as a therapeutic target against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Background

Coronavirus disease 2019 (COVID-19) causes significant morbidity and mortality worldwide and warrants the development of therapeutics against SARS-CoV-2. The authors of this study previously found that a calpain inhibitor molecule effectively inhibited SARS-CoV-2 by targeting her M.^Professional (major protease) A viral enzyme that is important for the processing of SARS-CoV-2 proteins.

About research

In the present study, researchers extended their previous analysis by investigating caplain-2 as a potential target for developing anti-SARS-CoV-2 agents.

The authors recently screened antiviral compounds using Vero E6 cells and recombinant SARS-CoV-2mNeonGreen virus.Several compounds showed anti-SARS-CoV-2 Effectiveness, and the top 18 hits were validated in this study. Validation experiments used a recombinant vesicular stomatitis virus (VSV) e-green fluorescent protein (eGFP) reporter virus encoding the spike (S) protein of native VSV-G or SARS-CoV-2.

To investigate whether MG132 compounds targeting host CPN2 can inhibit SARS-CoV-2, we used calpain inhibitor molecules such as ALLN, calpeptin, E-64d and calpain inhibitor III. These molecules are different because they inhibit calpains with varying specificities and target the calpain family.

To assess the involvement of CAPN2 in COVID-19, we tested lentivirus-promoted Cas9/CRISPR (clustered regularly spaced short palindromic repeats) among MA104 cells expressing angiotensin-converting enzyme 2 (ACE2). sequence) was used to perform gene knockout (KO) of the gene. Important for virus intrusion.

In addition, we performed western blot analysis to validate the CAPN2 KO efficiency. CAPN2 KO and wild-type (WT) cells were inoculated with SARS-CoV-2 S protein expressing a chimeric VSV (VSV-SARS-CoV-2).

A standard plaque assay of VSV-SARS-CoV-2 infection was performed to determine when CAPN2 exerted its proviral effect. KO and WT cells were inoculated with VSV-SARS-CoV-2 and levels of viral messenger ribonucleic acid (mRNA) at 1.0–6.9 hours post-infection (hpi) were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR).

Additionally, recombinant SARS-CoV-2 strains with S mutations, D614G, E484K and N501Y were tested. Single clone CAPN2 KO MA104 cells were generated and confirmed by Sanger sequencing. In addition, we performed a conventional His VSV-SARS-CoV-2 cold binding assay to investigate whether VSV-SARS-CoV-2 adsorption is negatively affected by the absence of caplain-2 and S by CAPN2. We assessed the potential for protein cleavage.

Co-transfection experiments were performed using ACE2-expressing human embryonic kidney (HEK) 293 cells and WA1 strain S protein to detect CAPN2, furin, or TMPRSS2 (transmembrane serine protease 2), a key protease, has been reported to cleave the S protein for efficient host entry. The team then collected cell lysates and quantified the intensity of the total S protein and the cleaved product, subunit 2 (S2), before assessing his ACE2 levels in CAPN2 KO and WT cells.

result

Calpain inhibitors (calpain inhibitor III, calpeptin, E-64d, and ALLN) inhibit VSV-SARS-CoV-2 at 50% effective inhibitory concentrations (EC₅₀) values ​​were <1.5 μM, but not Mpro, in contrast, calpain inhibitors showed no antiviral activity against the native protein of WT VSV. VSV-SARS-CoV-2 infection was significantly reduced among calpain-2 KO cells, and CAPN2 was critical for the early stages of SARS-CoV-2 proliferation. CAPN2 promoted SARS-CoV-2 binding to host cells.

Our findings showed that calpain inhibitor molecules target Mpro-independent pathways to effectively inhibit SARS-CoV-2 and calpain-2. This is a novel host factor that could potentially target the host cell invasion step to broaden the therapeutic landscape of COVID-19. Most antiviral compounds tested showed dose-dependent infection with VSV and VSV-SARS-CoV-2 inhibition among MA104 cells.

IMBX (3-isobutyl-1-methylxanthine), nigericin and Brefeldin A showed EC₅₀ Values ​​below 2.0 μM for infection with VSV and VSV-SARS-CoV-2.Nitazoxanide showed SARS-CoV-2 inhibition and MG132 was 100-fold selective in EC in activity against VSV and VSV-SARS-CoV-2₅₀ values ​​of 44 and 0.6 µM, respectively. Inhibitors other than calpeptin showed no cytotoxicity. VSV-SARS-CoV-2 mRNA was 4.0-fold lower in cells lacking CAPN2. The size of VSV-SARS-CoV-2 plaques was 1.0 mm in KO cells and 2.0 mm in WT cells. However, there was no significant difference in his VSV plaque size between KO and WT cells.

GFP signal from VSV-SARS-CoV-2 was reduced among CAPN2 knockout cells at 6 hpi, indicating that CAPN2 aided SARS-CoV-2 replication. We found significantly lower VSV-SARS-CoV-2 mRNA and S protein levels in KO cells compared to WT cells. Interestingly, a non-significantly lower level of mRNA was found in CAPN2 knockout cells. This indicates that her CAPN2 effect on SARS-CoV-2 probably depends on the nature of the S protein.

Furthermore, the levels of viral mRNA in KO and WT cells were similar in the presence of antibody incubation. Colocalization analysis of WGA (wheat germ agglutinin) and ACE2 showed significantly lower surface ACE2 levels in CAPN2 193 KO cells.

Conclusion

Overall, the study results indicated that calpain-2 positively regulates the presence of ACE2 on the cell surface, thereby improving S-mediated binding and infectivity of SARS-CoV-2. Therefore, CAPN2 can be viewed as a novel proviral factor that aids SARS-CoV-2 host cell entry.

*Important Notices

bioRxiv publishes non-peer-reviewed, preliminary scientific reports and should not be considered conclusive, to guide clinical practice/health-related actions, or to be treated as established information .