Characterization of Hydrophobic Interactions of SARS-CoV-2 and MERS-CoV Spike Protein Fusion Peptides Using Single Molecule Force Measurements

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide sequences within coronavirus (CoV) spike proteins. Within the fusion peptides of SARS-CoV-2 and MERS-CoV, a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. While a non-polar triad (LLF) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore if single molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask if sequence variations between FP1 from SARS and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 {+/-} 0.03 nN versus 0.68 {+/-} 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single residue diversity in viral fusion peptides, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts. SIGNIFICANCEFusion of coronaviruses (CoVs) and host cells is mediated by the insertion of the fusion peptide (FP) of the viral spike protein into the host cell membrane. Hydrophobic interactions between FPs with their host cell membranes regulate the viral membrane fusion process and are key to determining infection ability. However, it is not fully understood how the amino acid sequences in FPs mediate hydrophobic interactions. We use single-molecule force measurements to characterize hydrophobic interactions of FPs from SARS-CoV-2 and MERS-CoV. Our findings provide insight into the mechanisms by which the amino acid composition of FPs encodes hydrophobic interactions and their implications for fusion activity critical to the spread of infection.

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

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.

Spike protein cleavage-activation mediated by the SARS-CoV-2 P681R mutation: a case-study from its first appearance in variant of interest (VOI) A.23.1 identified in Uganda

Based on its predicted ability to affect transmissibility and pathogenesis, surveillance studies have highlighted the role of a specific mutation (P681R) in the S1/S2 furin cleavage site of the SARS-CoV-2 spike protein. Here we analyzed A.23.1, first identified in Uganda, as a P681R-containing virus several months prior to the emergence of B.1.617.2 (Delta variant). We performed assays using peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin compared to both an original B lineage (Wuhan-Hu1) and B.1.1.7 (Alpha variant). We also performed cell-cell fusion and functional infectivity assays using pseudotyped particles and observed an increase in activity for A.23.1 compared to an original B lineage spike. However, these changes in activity were not reproduced in the B lineage spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, this substitution needs to occur on the background of other spike protein changes to enable its functional consequences.

Functional evaluation of proteolytic activation for the SARS-CoV-2 variant B.1.1.7: role of the P681H mutation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern (VOC) first identified in the UK in late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7 or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that while the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage this does not significantly impact viral entry or cell-cell spread. HighlightsO_LISARS-CoV-2 B.1.1.7 VOC has a P681H mutation in the spike that is predicted to enhance viral infection C_LIO_LIP681H does not significantly impact furin cleavage, viral entry or cell-cell spread C_LIO_LIOther mutations in the SARS-CoV-2 B.1.1.7 VOC may account for increased infection rates C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/438731v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@c148d7org.highwire.dtl.DTLVardef@1954eeeorg.highwire.dtl.DTLVardef@171130dorg.highwire.dtl.DTLVardef@99bd45_HPS_FORMAT_FIGEXP M_FIG C_FIG

Proteolytic activation of the SARS-CoV-2 spike S1/S2 site: a re-evaluation of furin cleavage

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2 site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 pre-cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.

FDA approved calcium channel blockers inhibit SARS CoV 2 infectivity in epithelial lung cells

COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus (CoV)-2 (SARS-CoV-2). The virus is responsible for an ongoing pandemic and concomitant public health crisis around the world. While vaccine development is proving to be highly successful, parallel drug development approaches are also critical in the response to SARS-CoV-2 and other emerging viruses. Coronaviruses require Ca2+ ions for host cell entry and we have previously shown that Ca2+ modulates the interaction of the viral fusion peptide with host cell membranes. In an attempt to accelerate drug development, we tested a panel of L-type calcium channel blocker (CCB) drugs currently developed for other conditions, to determine whether they would inhibit SARS-CoV-2 infection in cell culture. All the CCBs tested showed varying degrees of inhibition, with felodipine and nifedipine strongly limiting SARS-CoV-2 entry and infection in epithelial lung cells at concentrations where cell toxicity was minimal. Further studies with pseudo-typed particles displaying the SARS-CoV-2 spike protein suggested that inhibition occurs at the level of virus entry. Overall, our data suggest that certain CCBs have potential to treat SARS-CoV-2 infections and are worthy of further examination for possible treatment of COVID-19.