Coronavirus-Induced Membrane Fusion Requires the Cysteine-Rich Domain in the Spike Protein

Chang, Kevin W.; Sheng, Yiwei; Gombold, James L.

doi:10.1006/viro.2000.0219

Cited by 58 publications

(87 citation statements)

References 76 publications

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“…This small region contains two cysteine residues conserved among all the coronaviruses. The homologous regions in SARS-CoV (LKGAC-SCGSCC) and MHV (FKKCGNCC) have been shown to play a role in cell-to-cell membrane fusion (Chang et al, 2000;Peti et al, 2005). Taken together, we suggest that these 11 residues contribute to membrane fusion during NL63 entry.…”

Section: Discussionmentioning

confidence: 69%

“…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%

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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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Section: Discussionmentioning

confidence: 69%

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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“…1A). This motif has been demonstrated to play an important role in cell-cell fusion during syncytium formation induced by the S protein, which might be associated with the palmitoylation of these cysteine residues (Bos et al, 1995;Chang et al, 2000;Thorp et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Partial deletion in the spike endodomain of mouse hepatitis virus decreases the cytopathic effect but maintains foreign protein expression in infected cells

Yang

Sun

Wang

et al. 2011

Journal of Virological Methods

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Mouse hepatitis virus (MHV) produces a series of subgenomic RNAs for viral protein expression. As a prototype coronavirus, MHV has been explored extensively and is often used to express foreign proteins. Previously, a 13-residue deletion in the MHV spike (S) protein endodomain was found to reduce syncytium formation dramatically while inhibiting virus replication slightly. In this study, the effects of the S mutation on MHV infectivity and foreign protein expression were further examined in rat or mouse L2, NIH/3T3 and Neuro-2a cells. The replacement of the MHV 2a/haemagglutinin-esterase gene with a membrane-anchored protein hook (HK) and replacement of gene 4 with EGFP did not change the adaptability and cytopathology of recombinant viruses in these cells. However, the cytopathic effect of the recombinants with the partial S deletion was reduced significantly in these cells. The replication and foreign protein expression of the S-mutated recombinants were found to be more efficient in L2 cells than in Neuro-2a and NIH/3T3 cells. Meanwhile, the distribution patterns of HK and EGFP expressed by the recombinant viruses were similar to those in cells transfected with a eukaryotic expression vector. These results suggest that the partial deletion in the S endodomain may increase the usefulness of MHV as a viral vector by attenuation and maintaining foreign protein expression.

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“…In addition to length, TM domains may exert a sequencedependent effect by altering lipid packing or membrane curvature within the donor lipid bilayer as part of the fusion process (8,9,29,34,49). In the case of p10, the small volume of the glycine residues and free mobility around the glycineglycine bonds could facilitate additional mobility of the fatty acyl groups of adjacent lipids.…”

Section: Discussionmentioning

confidence: 99%

Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

Shmulevitz

Salsman

Duncan

2003

J Virol

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Avian reovirus and Nelson Bay reovirus are two unusual nonenveloped viruses that induce extensive cell-cell fusion via expression of a small nonstructural protein, termed p10. We investigated the importance of the transmembrane domain, a conserved membrane-proximal dicysteine motif, and an endodomain basic region in the membrane fusion activity of p10. We now show that the p10 dicysteine motif is palmitoylated and that loss of palmitoylation correlates with a loss of fusion activity. Mutational and functional analyses also revealed that a triglycine motif within the transmembrane domain and the membrane-proximal basic region were essential for p10-mediated membrane fusion. Mutations in any of these three motifs did not influence events upstream of syncytium formation, such as p10 membrane association, protein topology, or surface expression, suggesting that these motifs are more intimately associated with the membrane fusion reaction. These results suggest that the rudimentary p10 fusion protein has evolved a mechanism of inducing membrane merger that is highly dependent on the specific interaction of several different motifs with donor membranes. In addition, cross-linking, coimmunoprecipitation, and complementation assays provided no evidence for p10 homo-or heteromultimer formation, suggesting that p10 may be the first example of a membrane fusion protein that does not form stable, higher-order multimers.Protein-mediated membrane fusion is an essential step in numerous cellular processes and in entry of enveloped viruses into cells (20,22,53). The viral fusion proteins involved in enveloped virus entry and virus-induced syncytium formation have been extensively characterized. Membrane fusion is dependent on extensive conformational changes in these complex, multimeric viral proteins that serve to regulate and drive the fusion reaction (46, 52). Much effort has been focused on defining the roles of specific fusion protein motifs in the membrane fusion reaction. Numerous studies have revealed the essential role that viral fusion peptides play in the membrane fusion reaction, serving to destabilize lateral lipid interactions and alter membrane curvature, leading to fusion pore formation (15,19,31,39,48). Studies on the influence of the transmembrane (TM) domains or endodomains of viral fusion proteins on membrane fusion, however, have yielded variable or contradictory results, and specific roles for these motifs remain unclear (1,9,34,35,38).Avian reovirus (ARV) and Nelson Bay reovirus (NBV) are two of only a limited number of nonenveloped viruses that induce cell-cell fusion and multinucleated-syncytium formation (12). Both viruses encode homologous 10-kDa proteins (p10) that, when expressed by themselves in transfected cells, induce extensive cell-cell fusion (45). These fusion-associated small transmembrane (FAST) proteins are significantly smaller than the enveloped virus fusion proteins, containing only 95 to 98 amino acids. The reovirus FAST proteins are also the only examples of nonstructural viral protei...

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Coronavirus-Induced Membrane Fusion Requires the Cysteine-Rich Domain in the Spike Protein

Cited by 58 publications

References 76 publications

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

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

Partial deletion in the spike endodomain of mouse hepatitis virus decreases the cytopathic effect but maintains foreign protein expression in infected cells

Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

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