FXa Cuts the SARS-CoV-2 Spike Protein And Blocks COVID-19 (Biology)

There have been several host serine proteases (SPs) which allows SARS-CoV-2 entry via cleavage of its spike (S) protein into functional S1 and S2 subunits. For instance, Furin cuts the S protein at the PRRAR (R-R-A-R685 ↓) site into S1 and S2 subunits at virus budding, while TMPRSS2 cleaves at the S2’ site (P-S-K R815↓) at virus entry, and both cleavages enhance efficiency of SARS-CoV-2 infection. But, we haven’t identified one which blocks SARS-CoV-2 entry.

Now, Wenjuan Dong and colleagues showed that FXa, a SP for blood coagulation, inhibits SARS-CoV-2 entry. Mechanistically, FXa binds to and cleaves S protein, which produced a different cleavage pattern than that of furin and TMPRSS2, and blocked S protein binding to ACE2.

To identify changes in SPs during SARS-CoV-2 infection, they examined their expression in lung samples from COVID-19 patients using an immunohistochemistry assay (IHC). They found that FX was significantly increased in the lungs of COVID-19 patients compared to non-COVID-19 donors. They also found that FX was also increased in the liver and serum, which is the source and carrier of FXa, in COVID-19 patients compared to non-COVID-19 donors.

In addition, to investigate the consequences of increased FXa during SARS-CoV-2 infection, they cloned FXa into the pCDH-mCherry vector and assessed its function using the vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeric virus. They found that, unlike pretreatment with furin, TMPRSS2, or trypsin, all of which increased VSV-SARS-CoV-2 infection, pre-treatment with FXa inhibited viral infection.

Furthermore, in order to determine if FXa blocks viral infection by targeting SARS-CoV-2 or host cells, they first constructed an FXa-Fc fusion protein expression plasmid and purified the protein from Chinese hamster ovary (CHO) cells. Then, they co-incubated VSV-SARS-CoV-2 with or without FXa-Fc fusion protein in vitro for 1 hour before adding the mixture into MA104 cells. Finally, they examined the rate of infection at the indicated timepoints and found that FXa could block viral infection by targeting SARS-CoV-2. However, they have been unable to identify the exact mechanism through which the FXa blocks entry of SARS-CoV-2 into host cells.

“We found that, FXa blocks the B.1.1.7 variant infection less efficiently compared to the Wild type SARS-CoV-2 due to different binding affinity to the corresponding S protein.”

Finally, COVID-19 patients with an increased risk of thrombosis are treated with direct FXa inhibitors (e.g., rivaroxaban) or indirect inhibitors (e.g., fondaparinux). They therefore examined if rivaroxaban (RIVA) or fondaparinux (FONDA) affect the anti-viral activity of FXa. They found that, neither RIVA nor FONDA blocked the binding of FXa to S protein, and neither drug alone had any effect on VSV-SARS-CoV-2 infectivity; however, the direct FXa inhibitor RIVA blocked FXa-induced anti-viral activity, whereas the indirect FXa inhibitor FONDA did not.

“We identify a new mechanism of anti-viral defense in human and prove its importance in a transgenic animal model that mimics the human disease. Our work would suggest that future studies should examine the quality and quantity of FXa enzymatic activity against the SARS-CoV- 2 S protein to determine if it can improve our understanding of who may be most susceptible to SARS-CoV-2 infection and who might be treated with an FXa indirect anti-coagulant inhibitor.”

— concluded authors of the study

Featured image: Schema of the cleavage sites for furin, TMPRSS2 and FXa on the full- length S protein. © Dong et al.

Reference: Jianhua Yu, Wenjuan Dong, Jing Wang, Lei Tian, Jianying Zhang, Heather L. Mead, Sierra Jaramillo, Aimin Li, Ross Zumwalt, Sean P. J. Whelan, Erik Settles, Paul Keim, Bridget M. Barker, Michael Caligiuri, “FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant”, bioRxiv 2021.06.07.447437; doi: https://doi.org/10.1101/2021.06.07.447437

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