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National Institute of General Medical Sciences
	National Biomedical Resource for Advanced ESR Spectroscopy

ACERT's Service and Collaborative Projects

SARS-CoV-2 Fusion Peptide Has a Greater Membrane Perturbation Effect than SARS-CoV with Highly Specific Dependence on Ca²⁺

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein, which is structurally characterized as a class I viral fusion protein, within the same group as influenza virus and HIV. In our previous ESR studies, the local membrane ordering effect of various viruses including SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2' cleavage site is the bona fide SARS-1 fusion peptide (FP), and proposed an extended fusion "platform" model. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. We found by ESR that although there is a difference of only three residues, the SARS-2 FP induces even greater membrane ordering than the SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca²⁺, but that of SARS-2 shows a greater response to the presence of Ca²⁺. The SARS-2 FP binds two Ca²⁺ ions, one each in its FP1 and FP2 domains, as does SARS-1 FP, but the cooperativity of the two Ca²⁺ binding sites of SARS-2 is greater. This Ca²⁺ dependence by the SARS-2 FP is very ion-specific. These results show that Ca²⁺ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca²⁺ channel, and also repurpose already-approved drugs for the ongoing COVID-19 pandemic.

Publication: J. Mol. Biol. 433, 166946 (2021); PMC7969826.

Plots of local order parameters obtained by ESR of DPPTC (A), 5PC (B), 10PC (C) and 14PC(D) versus peptide:lipid ratio (P/L ratio) of SARS-2 FP, SARS FP and MERS FP in POPC/POPG/Chol=3/1/1 MLVs in buffer at pH5 with 150 mM NaCl at 25°C. Black, SARS-2 FP with 1 mM Ca²⁺ and at pH 5; red, SARS-2 FP with 1 mM EGTA; blue, SARS FP with 1 mM Ca²⁺; green, MERS FP with 1 mM Ca²⁺. (E). Plots of local order parameters of DPPTC versus P/L ratio as in A-D with pH indicated. Black, SARS-2 FP at pH 5; red, SARS-2 FP triple mutant at pH 5; blue, SARS-2 FP at pH 7; and green, a peptide with shuffled sequence of SARS-2 FP at pH 5. (F) Plots of local order parameters of DPPTC with and without 1% peptide binding (ΔS₀) versus Ca²⁺ as in A-D. Black, SARS-2 FP; blue, SARS FP and green, MERS FP.

Alex Liqi Lai and Jack H. Freed (ACERT)

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ACERT is supported by grant 1R24GM146107 from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

National Biomedical Resource for Advanced ESR Spectroscopy

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National Institute of
General Medical Sciences