SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion.
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ABSTRACT:
SUBMITTER: Madu IG
PROVIDER: S-EPMC3594805 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The β-Coronavirus mouse hepatitis virus (MHV-A59)-RSA59 has a patent stretch of fusion peptide (FP) containing two consecutive central prolines (PP) in the S2 domain of the Spike protein. Our previous studies compared the PP-containing fusogenic-demyelinating strain RSA59(PP) to its one proline-deleted mutant strain RSA59(P) and one proline-containing non-fusogenic non-demyelinating parental strain RSMHV2(P) to its one proline inserted mutant strain RSMHV2(PP). These studies highlighted the crucial role of PP in fusogenicity, hepato-neuropathogenesis, and demyelination. Computational studies combined with biophysical data indicate that PP at the center of the FP provides local rigidity while imparting global fluctuation to the Spike protein that enhances the fusogenic properties of RSA59(PP) and RSMHV2(PP). To elaborate on the understanding of the role of PP in the FP of MHV, the differential neuroglial tropism of the PP and P mutant strains was investigated. Comparative studies demonstrated that PP significantly enhances the viral tropism for neurons, microglia, and oligodendrocytes. PP, however, is not essential for viral tropism for either astroglial or oligodendroglial precursors or the infection of meningeal fibroblasts in the blood-brain and blood-CSF barriers. PP in the fusion domain is critical for promoting gliopathy, making it a potential region for designing antivirals for neuro-COVID therapy.
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Project description:Continuously emerging highly pathogenic human coronaviruses (HCoVs) remain a major threat to human health, as illustrated in past SARS-CoV and MERS-CoV outbreaks. The development of a drug with broad-spectrum HCoV inhibitory activity would address this urgent unmet medical need. Although previous studies have suggested that the HR1 of HCoV spike (S) protein is an important target site for inhibition against specific HCoVs, whether this conserved region could serve as a target for the development of broad-spectrum pan-CoV inhibitor remains controversial. Here, we found that peptide OC43-HR2P, derived from the HR2 domain of HCoV-OC43, exhibited broad fusion inhibitory activity against multiple HCoVs. EK1, the optimized form of OC43-HR2P, showed substantially improved pan-CoV fusion inhibitory activity and pharmaceutical properties. Crystal structures indicated that EK1 can form a stable six-helix bundle structure with both short α-HCoV and long β-HCoV HR1s, further supporting the role of HR1 region as a viable pan-CoV target site.
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Project description:The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here we report the cryoEM structure of a coronavirus S glycoprotein in the postfusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the prefusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochemically characterized the molecular events associated with refolding of the metastable prefusion S glycoprotein to the postfusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The observed similarity between postfusion coronavirus S and paramyxovirus F structures demonstrates that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.
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Project description:A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre- and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses.IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre- to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.
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Project description:The SARS coronavirus (SARS-CoV) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. Previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. In this study we have produced purified, irradiated SARS-CoV virions that retain their morphology, and are fusogenic in cell culture. We used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ACE2). We have shown that ACE2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. Furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ACE2 molecules. The SARS-CoV spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis.
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Project description:Proteolytic priming is a common method of controlling the activation of membrane fusion mediated by viral glycoproteins. The severe acute respiratory syndrome coronavirus spike protein (SARS-CoV S) can be primed by a variety of host cell proteases, with proteolytic cleavage occurring both as the S1/S2 boundary and adjacent to a fusion peptide in the S2 domain. Here, we studied the priming of SARS-CoV S by elastase and show an important role for residue Thr(795) in the S2 domain. A series of alanine mutants were generated in the vicinity of the S2 cleavage site, with the goal of examining elastase-mediated cleavage within S2. Both proteolytic cleavage and fusion activation were modulated by altering the cleavage site position. We propose a novel mechanism whereby SARS-CoV fusion protein function can be controlled by spatial regulation of the proteolytic priming site, with important implications for viral pathogenesis.
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Project description:The S1 and S2 subunits of the spike glycoprotein of the coronavirus which is responsible for the severe acute respiratory syndrome (SARS) have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures. An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the genomic organisation similarity generally exhibited by coronaviruses. The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein. The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity.
| S-EPMC7110993 | biostudies-literature
Project description:Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation=0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections.
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Project description:The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941-50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.
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Project description:Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel β-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human β-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human β-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.
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