The interaction of calcium ions with specific residues in the SARS-CoV fusion peptide and the regulation of viral infectivity

ABSTRACT

In coronaviruses, the fusion peptide (FP) is situated within the membrane fusion domain of the spike protein, becoming exposed following proteolytic cleavages at the S1/S2 and S2 sites. After receptor binding-induced conformational changes, the FP penetrates the host cell membrane and mediates membrane fusion. Previous work has revealed the importance of calcium for SARS-CoV-1 FP structural stability and host membrane insertion. In this follow-up study, we systematically introduced charge-neutralizing alanine mutations in the negatively charged amino acids within the SARS-CoV-1 fusion peptide (E801A, D802A, D812A, E821A, D825A, D830A) to identify residues that likely bind to calcium. We assayed fusion competency by performing a syncytia-formation assay in VeroE6 cells and infectivity using pseudoparticles. The loss of single negatively charged residues D812 or D830 greatly reduced syncytia formation and produced noninfectious pseudoparticles. Furthermore, we observed a calcium-dependent decrease in the infectivity of the D825A and D830A pseudoparticles, as well as fewer syncytia in the cells expressing E821A/D825A double mutant FP. To clarify which residue pairs in the FP are most likely to bind calcium and promote host membrane insertion, we carried out molecular dynamics (MD) simulations of the various FP constructs. From our modeling, residue E801 is predicted to pair with either D830 or D802 to coordinate one calcium; a second calcium ion likely pairs residue E821 with either D812 or D825. We propose a model of bimodal calcium binding in the FP1 and FP2 domains, which anchors the SARS-CoV-1 FP in the host cell membrane to promote membrane insertion and fusion.