The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity

Bos, Evelyne; Luytjes, Willem; Spaan, Willy J. M.

doi:10.1128/jvi.71.12.9427-9433.1997

Cited by 33 publications

(32 citation statements)

References 53 publications

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“…We selected this region of the MHV-A59 S protein for our initial studies and created a plasmid, designated pCAGGS-S (546-548), for expressing a mutant S protein in which residues R546/C547/Q548 were changed to alanine. To determine the effect that this mutation had on the overall conformation of the protein we compared its expression and its immunoreactivity with two different conformationally sensitive anti-S monoclonal antibodies, known to neutralize MHV-A59 infectivity and block infection with virus like particles containing the MHV-A59 structural proteins (Gilmore et al, 1987;Bos et al, 1997) compared to that of wild type S protein expressed in the same vector. Replicate cultures of 293 T cells were transfected with pCAGGS-S or pCAGGS-S (546-548) and after 24 h post transfection the cells were metabolically labeled with 35 S (cysteine and methionine) for 9 h, harvested, cytoplasmic extracts prepared and then immunoprecipitated with the anti-spike monoclonal neutralizing antibodies A2.1 and A2.3 (Gilmore et al, 1987) or with a polyclonal rabbit antispike antibody, B46 (kindly provided by Dr. John Fleming and Dr. K. Holmes, respectively).…”

Section: Alanine Substitution Of Cysteine 547 Facilitates Partial Recmentioning

confidence: 99%

Identification of novel functional regions within the spike glycoprotein of MHV-A59 based on a bioinformatics approach

2014

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Mouse Hepatitis Virus (MHV) is a single-stranded positive sense RNA virus with the ability to promote acute and chronic diseases in mice. The MHV spike protein (S) is a major virulence determinant which in addition to binding to cellular receptors to mediate cell entry and facilitate virus spread to adjacent cells by cell-cell fusion, also is a molecular mimic of the FcγRII receptor. This molecular mimicry of FcγRII by the MHV S protein is also exhibited by other lineage 2a betacoronaviruses, with the exception of the human coronavirus HCoV-OC43. In this work we undertook a mutational analysis to attempt to identify specific amino acid sequences within the spike glycoprotein crucial for molecular mimicry of FcγRII. Although we were unsuccessful in isolating mutant viruses which were specifically defective in that property, we identified several mutations with interesting phenotypes. Mutation of the cysteine in position 547 to alanine and alanine replacements at residues 581–586 was lethal. Replacing proline 939 with the corresponding HCoV-OC43 residue, leucine, decreased the ability MHV to induce cell-cell fusion, providing experimental support for an earlier proposal that residues 929–944 make up the fusion peptide of the MHV S protein.

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Section: Alanine Substitution Of Cysteine 547 Facilitates Partial Recmentioning

confidence: 99%

Identification of novel functional regions within the spike glycoprotein of MHV-A59 based on a bioinformatics approach

2014

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“…The requirement for proteolytic maturation among the normally cleaved S glycoproteins of the group 2 CoV species is likewise ambiguous. In the MHV glycoprotein, mutations that alter the furin protease recognition sequence (R-X-R/K-R↓; (Molloy et al, 1999;Rockwell et al, 2002) and references therein) and prevent cleavage generally diminish cell-cell fusion activity (Bos et al, 1995;de Haan et al, 2004;Sturman et al, 1985;Taguchi, 1993;Yamada et al, 1998), yet do not markedly affect viral infectivity or pathogenesis (Bos et al, 1997;de Haan et al, 2004;Hingley et al, 2002). By contrast, MHV infectivity can be enhanced by exogenous treatment with trypsin (Sturman et al, 1985), raising the possibility that proteases that recognize single basic residues may be important.…”

Section: Introductionmentioning

confidence: 99%

Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell–cell fusion but does not affect virion entry

2006

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The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctional region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.

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“…The S glycoprotein is primarily responsible for entry of all coronaviruses into susceptible cells through binding to specific receptors on cells and mediating subsequent virus-cell fusion (Cavanagh, 1995). The S glycoprotein specified by mouse hepatitis virus (MHV) is cleaved into S1 and S2 subunits, although cleavage is not necessarily required for virus-cell fusion (Bos et al, 1997;Gombold et al, 1993;Stauber et al, 1993). Similarly, the SARS-CoV S glycoprotein seems to be cleaved into S1 and S2 subunits in Vero-E6-infected cells , while it is not known whether this cleavage affects S-mediated cell fusion.…”

Section: Introductionmentioning

confidence: 99%

Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

et al. 2005

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The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion.

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The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity

Cited by 33 publications

References 53 publications

Identification of novel functional regions within the spike glycoprotein of MHV-A59 based on a bioinformatics approach

Identification of novel functional regions within the spike glycoprotein of MHV-A59 based on a bioinformatics approach

Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell–cell fusion but does not affect virion entry

Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

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