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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-329644

ABSTRACT

Viral envelope fusion with the host cell membrane is dependent on a specific viral fusion peptide (FP) or loop, which becomes exposed during virus entry to drive the process of membrane fusion. In coronaviruses, the FP is a highly conserved domain that sits in the center of spike protein and in SARS-CoV, is adjacent to the S2’ proteolytic cleavage site. This peptide contains a hydrophobic LLF motif, as well as several conserved negatively charged amino acids that interact with Ca2+ ions to promote membrane fusion. In this work we perform a systematic mutagenesis study of the negatively charged amino acids within the SARS-CoV fusion peptide (FP1/FP2) and combine this with molecular dynamics simulations to define the membrane interactions that regulate virus infectivity. We show that the E801/D802 amino acid pair in the SARS-CoV FP is predicted to bind to one Ca 2+ ion to promote FP-membrane interaction, with a second Ca 2+ ion likely pairing residue D812 with either E821 or D825. The D812/D821 residue pair promotes membrane interaction, whereas the D821/D825 is inhibitory to membrane insertion. Taken together, our results demonstrate the dynamic nature of the coronavirus FP region that likely facilitates its interactions with and insertion into the host cell membrane. Author Summary Coronaviruses have reemerged as a highly pathogenic virus family through the rise of SARS-CoV, MERS-CoV, and more recently, SARS-CoV-2. As more transmissible variants of SARS-CoV-2 arise, it is imperative that we understand the mechanisms of CoV viral entry to enable the development of effective therapeutics. Recent reviews have suggested the repurposing of FDA-approved calcium channel blockers to treat infection by coronaviruses;however, calcium’s method of action on viral-host cell fusion events is unknown. We have found that increased calcium availability leads to increased viral infection across the CoV family, suggesting that calcium is involved in mediating the interaction between the viral fusion peptide and the host cell membrane. As such, we hypothesize that the highly conserved fusion peptide interacts directly with calcium and this interaction is required for viral entry and infection. Through mutagenesis studies of specific negatively charged residues in the fusion peptide, we have identified residues that impact viral infectivity. We have also compared the infectivity of wild-type and mutant CoV pseudoparticles in calcium-rich or -depleted environments using chelating drugs. Our data mirrors the residue coordination observed SARS-CoV-2, as both between SARS-CoV and SARS-CoV-2 FPs bind to two calcium ions. These results demonstrate the importance of Ca 2+ for CoV FP function during viral entry and opens the possibility of utilizing FDA-approved calcium-blocking drugs as a treatment for COVID-19.

2.
J Virol ; 94(13)2020 06 16.
Article in English | MEDLINE | ID: covidwho-1583223

ABSTRACT

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.


Subject(s)
Calcium/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Host-Pathogen Interactions , Ions/metabolism , Membrane Fusion , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Internalization , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Chlorocebus aethiops , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Models, Molecular , Mutation , Protein Binding , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Vero Cells , Virulence , Virus Assembly
3.
ACS Infect Dis ; 7(10): 2807-2815, 2021 10 08.
Article in English | MEDLINE | ID: covidwho-1402020

ABSTRACT

COVID-19 is caused by a novel coronavirus, the 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 repurposing, 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 pseudotyped 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 the potential to treat SARS-CoV-2 infections and are worthy of further examination for possible treatment of COVID-19.


Subject(s)
COVID-19 , Pharmaceutical Preparations , Calcium Channels, L-Type , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Virus Internalization
4.
ACS Infect Dis ; 7(2): 264-272, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1023823

ABSTRACT

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 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.


Subject(s)
COVID-19/virology , Furin/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Models, Molecular , Peptide Hydrolases/chemistry , Proteolysis , SARS-CoV-2/chemistry , Vero Cells , Virus Internalization
5.
Antiviral Res ; 178: 104792, 2020 06.
Article in English | MEDLINE | ID: covidwho-34819

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapies against the causative agent, SARS-CoV-2, and also against other pathogenic coronaviruses (CoV) that have emerged in the past or might appear in future. Researchers are therefore focusing on steps in the CoV replication cycle that may be vulnerable to inhibition by broad-spectrum or specific antiviral agents. The conserved nature of the fusion domain and mechanism across the CoV family make it a valuable target to elucidate and develop pan-CoV therapeutics. In this article, we review the role of the CoV spike protein in mediating fusion of the viral and host cell membranes, summarizing the results of research on SARS-CoV, MERS-CoV, and recent peer-reviewed studies of SARS-CoV-2, and suggest that the fusion mechanism be investigated as a potential antiviral target. We also provide a supplemental file containing background information on the biology, epidemiology, and clinical features of all human-infecting coronaviruses, along with a phylogenetic tree of these coronaviruses.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Membrane Fusion/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Animals , Betacoronavirus/physiology , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Humans , Molecular Targeted Therapy , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
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