Abstract
The spike (S) glycoprotein of the SARS-CoV-2 virus binds to the host cell receptor and promotes the virus’s entry into the target host cell. This interaction is primed by host cell proteases like furin and TMPRSS2, which act at the S1/S2 and S2´ cleavage sites, respectively. Both cleavage sites have serine or proline residues flanking either the single or polybasic region and were found to be conserved in coronaviruses. Unravelling the effects of these conserved residues on the virus entry and infectivity might facilitate the development of novel therapeutics. Here, we have investigated the role of the conserved serine and proline residues in the SARS-CoV-2 spike mediated entry, fusogenicity, and viral infectivity by using the HIV-1/spike-based pseudovirus system. A conserved serine residue mutation to alanine (S2´S-A) at the S2´ cleavage site resulted in the complete loss of spike cleavage. Exogenous treatment with trypsin or overexpression of TMPRSS2 protease could not rescue the loss of spike cleavage and biological activity. The S2´S-A mutant showed no significant responses against E-64d, TMPRSS2 or other relevant inhibitors. Taken together, serine at the S2´ site in the spike protein was indispensable for spike protein cleavage and virus infectivity. Thus, novel interventions targeting the conserved serine at the S2´ cleavage site should be explored to reduce severe disease caused by SARS-CoV-2-and novel emerging variants.
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The data supporting the findings of this study are available from the corresponding author upon request.
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Acknowledgements
We sincerely thank Prof Pramod Kumar Garg, Executive Director, THSTI for full support and valuable input and guidance. We thank Prof. S Pöhlmann, Infection Biology Unit, Göttingen, Germany for providing ACE2-Fc plasmids as a kind gift. We thank Dr. B Graham (VRC/NIAID/NIH) for providing us with the spike construct (SARS-2-CoV S 2P). The following reagent was obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK293T) Expressing Human Angiotensin-Converting Enzyme 2, HEK293T-hACE2 Cell Line, NR-52511.The TMPRSS2 plasmid was a gift from Roger Reeves (Addgene plasmid # 53887).
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This work was supported by the Department of Biotechnology (DBT), Govt. of India and THSTI core funding.
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SS and RK conceived the project. RK, BL, GK, VM and A.G conducted most of the experiments. DS and SA performed the molecular dynamic simulation study. SS and RK analyzed the data. SS wrote the original manuscript; RK and BL edited the manuscript.
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Khatri, R., Lohiya, B., Kaur, G. et al. Understanding the role of conserved proline and serine residues in the SARS-CoV-2 spike cleavage sites in the virus entry, fusion, and infectivity. 3 Biotech 13, 323 (2023). https://doi.org/10.1007/s13205-023-03749-y
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DOI: https://doi.org/10.1007/s13205-023-03749-y