Characterization of the Prefusion and Transition States of Severe Acute Respiratory Syndrome Coronavirus S2-HR2

McReynolds, Susanna; Jiang, Sanyuan; Guo, Ying; Celigoy, Jessica; Schar, Christine R.; Rong, Lijun; Caffrey, Michael

doi:10.1021/bi800622t

Cited by 21 publications

(41 citation statements)

References 34 publications

(90 reference statements)

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“…Given that the endodomain Cys 3 Ala mutations progressively extend the HR2-sensitive stage, we suggest that the absence of these cysteines-palmitates raises an activation energy barrier between the HR2-sensitive and 6-HB stage. It was recently discovered that the SARS-CoV HR2 regions exist in a monomer-trimer equilibrium (79). The idea is that the equilibrium has to be shifted toward monomers, so that separated HR2 helices can each invert relative to HR1 and attach in antiparallel fashion onto the HR1 trimers (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…6). Given that the HR2 regions in isolation can stick together into trimers (79), the role of the endodomain cysteines-palmitates could be to anchor the transmembrane spans such that a separation of HR2 monomers is maintained in the native S structure. This prevention of HR2 trimerization in the native structure would then allow membrane fusion to occur in a timely fashion.…”

Section: Discussionmentioning

confidence: 99%

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Role of Spike Protein Endodomains in Regulating Coronavirus Entry

Shulla

Gallagher

2009

Journal of Biological Chemistry

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Enveloped viruses enter cells by viral glycoprotein-mediated binding to host cells and subsequent fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated membrane-associated tails. Palmitate adducts are generally required for protein-mediated fusions, but their precise roles in the process are unclear. To obtain additional insights into the S-mediated membrane fusion process, we focused on these acylated carboxyl-terminal intravirion tails. Substituting alanines for the cysteines that are subject to palmitoylation had effects on both S incorporation into virions and S-mediated membrane fusions. In specifically dissecting the effects of endodomain mutations on the fusion process, we used antiviral heptad repeat peptides that bind only to folding intermediates in the S-mediated fusion process and found that mutants lacking three palmitoylated cysteines remained in transitional folding states nearly 10 times longer than native S proteins. This slower refolding was also reflected in the paucity of postfusion six-helix bundle configurations among the mutant S proteins. Viruses with fewer palmitoylated S protein cysteines entered cells slowly and had reduced specific infectivities. These findings indicate that lipid adducts anchoring S proteins into virus membranes are necessary for the rapid, productive S protein refolding events that culminate in membrane fusions. These studies reveal a previously unappreciated role for covalently attached lipids on the endodomains of viral proteins eliciting membrane fusion reactions.Biological membranes are configured in large part by protein-mediated fission and fusion reactions. Enveloped viruses can reveal the principles of these processes because their assembly and budding from infected cells requires membrane fissions, and their entry into susceptible cells depends on membrane fusions. Glycoproteins extending from virion surfaces mediate the fusion process. These specialized integral membrane proteins are in metastable high energy configurations on virus surfaces, and they drive coalescence of opposing virus and cell membranes by undergoing a series of energy-releasing unfolding and refolding events (1). The structural rearrangements are triggered by virus binding to cellular receptors (2) and by the acidic, proteolytic environments encountered after viruses are endocytosed (3-5). These reactions begin with an unfolding process that reveals hydrophobic fusion peptides (FPs) 2 that dagger into cellular membranes. This is then followed by a refolding process that, in analogy to a closing hairpin, brings FPs and associated cellular membranes toward the virion membranes, driving formation of a lipid stalk connecting the opposing outer membrane leaflets (6) and culminating in complete cell-virion membrane coalescence (7,8). For viral fusion proteins in the so-called "class I" category, the arms...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Role of Spike Protein Endodomains in Regulating Coronavirus Entry

Shulla

Gallagher

2009

Journal of Biological Chemistry

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“…Recently, S2-HR2 has been shown to be in a dynamic equilibrium between a structured coiled-coil trimer, which is thought to represent the prefusion state, and unstructured monomer, which is thought to represent a metastable transition state. [20] In the present work, we present the detailed characterization of the dynamic properties of SARS-CoV S2-HR2 in the prefusion and transition states using NMR relaxation. We will discuss the importance of considering the S2-HR2 dynamic properties in the viral entry model, as well as in the design of future envelope-based antivirals.…”

mentioning

confidence: 99%

“…[21] The backbone assignments at 25°C in the presence and absence of 30% TFE have been reported elsewhere. [13,20] The backbone assignments of S2-HR2 at 45°C were determined from a series of the standard triple resonance experiments. [13] The 15 N R z (1/ T 1 ), R xy (1/ T 2 ) and HNOE values were measured by standard pulse sequences.…”

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confidence: 99%

“…[13] On the other hand, in the absence of co-solvent, at 25°C S2-HR2 is in equilibrium between unstructured monomer and a structured trimer, which is thought to represent an equilibrium between the prefusion and transition states. [20] Moreover, in the absence of co-solvent, S2-HR2 exhibits a melting temperature of ~35°C and is completely unstructured at 45°C. [20] To gain insight into the dynamic properties of the prefusion and transition states, we characterized the dynamic properties of S2-HR2 under 2 experimental conditions: 10 mM NaPO 4 /pH 7.0, 30% TFE at 25°C (prefusion state) and 10 mM NaPO 4 /pH 7.0 at 45°C (transition state).…”

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confidence: 99%

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Dynamics of SARS-coronavirus HR2 domain in the prefusion and transition states

McReynolds

Jiang

Rong

et al. 2009

Journal of Magnetic Resonance

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The envelope glycoproteins S1 and S2 of severe acute respiratory syndrome coronavirus (SARS-CoV) mediate viral entry by conformational change from a prefusion state to a postfusion state that enables fusion of the viral and target membranes. In this work we present the characterization of the dynamic properties of the SARS-CoV S2-HR2 domain (residues 1141–1193 of S) in the prefusion and newly discovered transition states by NMR 15 N relaxation studies. The dynamic properties of the different states, which are stabilized under different experimental conditions, extend the current model of viral membrane fusion and give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses such as HIV.

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SARS‐CoV heptad repeat 2 is a trimer of parallel helices

Celigoy¹,

Ramirez²,

Caffrey³

2011

Protein Science

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In severe acute respiratory syndrome coronavirus, the envelope heptad repeat 2 (HR2) plays a critical role in viral entry. Moreover, HR2 is both the target for novel antiviral therapies and, as an isolated peptide, presents a potential antiviral therapeutic. The structure of HR2, as determined by NMR spectroscopy in the presence of the co-solvent trifluoroethanol (TFE), is a trimer of parallel helices, whereas the structure of HR2, as determined by X-ray crystallography, is a tetramer of anti-parallel helices. In this work, we added a nitroxide spin label to the N-terminal region of HR2 and used paramagnetic relaxation enhancement to assess the orientation of the HR2 helices under different solution conditions. We find that the relaxation effects are consistent with an orientation corresponding to a trimer of parallel helices in both the presence and absence of TFE. This work suggests that the different orientation and oligomerization states observed by NMR and X-ray are due to the 11 additional residues present at the N-terminus of the NMR construct.

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Characterization of the Prefusion and Transition States of Severe Acute Respiratory Syndrome Coronavirus S2-HR2

Cited by 21 publications

References 34 publications

Role of Spike Protein Endodomains in Regulating Coronavirus Entry

Role of Spike Protein Endodomains in Regulating Coronavirus Entry

Dynamics of SARS-coronavirus HR2 domain in the prefusion and transition states

SARS‐CoV heptad repeat 2 is a trimer of parallel helices

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