Spike protein assembly into the coronavirion: exploring the limits of its sequence requirements

Bosch, Berend Jan; Haan, Cornelis A. M. de; Smits, Saskia L.; Rottier, Peter J. M.

doi:10.1016/j.virol.2005.02.001

Cited by 52 publications

(68 citation statements)

References 66 publications

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“…25,27,40 In regard to minimize cross-reactivity, we incorporated spike (S) protein as an additional marker which exhibits least exhibition of sequence conservation among coronavirus proteins. 3,39 The results of WB analysis support the specificities of both the N and S-based ELISA. No seropositive serum, above the cutoff values, were failed by WB tested by its target antigen.…”

Section: Discussionsupporting

confidence: 55%

Examination of seroprevalence of coronavirus HKU1 infection with S protein-based ELISA and neutralization assay against viral spike pseudotyped virus

Chan

Tse

Wong

et al. 2009

Journal of Clinical Virology

102

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Section: Discussionsupporting

confidence: 55%

Examination of seroprevalence of coronavirus HKU1 infection with S protein-based ELISA and neutralization assay against viral spike pseudotyped virus

Chan

Tse

Wong

et al. 2009

Journal of Clinical Virology

102

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“…The exact boundaries of transmembrane (TM) domains of coronaviruses have not yet been experimentally specified. Different predictions include or exclude part of the cysteine-rich domains as part of the TM domain (Bosch et al, 2005;Broer et al, 2006;Chang et al, 2000;Godeke et al, 2000;Petit et al, 2007;Thorp et al, 2006;Ye et al, 2004;Zheng et al, 2006). According to TMpred, the strongly preferred TM model of NL63 S protein predicts a TM to be located between residues 1299 and 1316 (VWLIISVVFVVLLSLLVF), which does not include any cysteine-rich domains.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry

Lin

Yan

et al. 2011

Virus Research

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The spike (S) protein of human coronavirus NL63 (HCoV-NL63) mediates both cell attachment by binding to its receptor hACE2 and membrane fusion during virus entry. We have previously identified the receptor-binding domain (RBD) and residues important for RBD-hACE2 association. Here, we further characterized the S protein by investigating the roles of the cytoplasmic tail and 19 residues located in the RBD in protein accumulation, receptor binding, and pseudotype virus entry. For these purposes, we first identified an entry-efficient S gene template from a pool of gene variants and used it as a backbone to generate a series of cytoplasmic tail deletion and single residue substitution mutants. Our results showed that: (i) deletion of 18aa from the C-terminus enhanced the S protein accumulation and virus entry, which might be due to the deletion of intracellular retention signals; (ii) further deletion to residue 29 also enhanced the amount of S protein on the cell surface and in virion, but reduced virus entry by 25%, suggesting that residues 19-29 contributes to membrane fusion; (iii) a 29aa-deletion mutant had a defect in anchoring on the plasma membrane, which led to a dramatic decrease of S protein in virion and virus entry; (iv) a total of 15 residues (Y498, V499, V531, G534, G537, D538, S540, G575, S576, E582, W585, Y590, T591, V593 and G594) within RBD were important for receptor binding and virus entry. They probably form three receptor binding motifs, and the third motif is conserved between NL63 and SARS-CoV.

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“…Indeed, each S trimer would add 27 16-carbon acyl chain lipids to the intravirion membrane leaflet. Several reports evaluating truncated coronavirus S proteins missing part or all of these acylated tails have provided valuable data on the minimal tail lengths required to preserve biological function (28,30,67,68). We used a more subtle approach to evaluate tail activities by substituting one or more of the nine cysteines in the palmitoylation motif with alanines.…”

Section: Discussionmentioning

confidence: 99%

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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Spike protein assembly into the coronavirion: exploring the limits of its sequence requirements

Cited by 52 publications

References 66 publications

Examination of seroprevalence of coronavirus HKU1 infection with S protein-based ELISA and neutralization assay against viral spike pseudotyped virus

Examination of seroprevalence of coronavirus HKU1 infection with S protein-based ELISA and neutralization assay against viral spike pseudotyped virus

Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry

Role of Spike Protein Endodomains in Regulating Coronavirus Entry

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