Molecular cloning and expression of a spike protein of neurovirulent murine coronavirus JHMV variant c1-2

Taguchi, Fumihiro; Ikeda, Toshio; Shida, Hisatoshi

doi:10.1099/0022-1317-73-5-1065

Cited by 45 publications

(52 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, the S1 subunit is relatively variable among MHV; deletions and mutations often occur in the hypervariable region (22,31,62). When compared with the S protein of MHV-4, there are three deletions of one, three, and eight amino acid residues in Yamada's MHV-2 strain (31,42,53) and a large deletion of 141 and 153 amino acid residues in the fusion-positive MHV-JHM strain and our fusion-negative MHV-2 strain, respectively, in the hypervariable region of the S1 subunit.…”

Section: Discussionmentioning

confidence: 99%

A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

Tsai

Chang

1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

The spike (S) glycoprotein of mouse hepatitis virus (MHV) plays a major role in the viral pathogenesis. It is often processed into the N-terminal S1 and the C-terminal S2 subunits that were evidently important for binding to cell receptor and inducing cell-cell fusion, respectively. As a consequence of cell-cell fusion, most of the naturally occurring infections of MHV are associated with syncytia formation. So far, only MHV-2 was identified to be fusion-negative. In this study, the S gene of MHV-2 was molecularly cloned, and the nucleotide sequence was determined. The MHV-2 S protein lacks a 12-amino acid stretch in the S1 hypervariable region Mouse hepatitis virus (MHV)1 is a member of the Coronaviridae family that causes inapparent enteric and respiratory infection, hepatitis, and acute and chronic demyelinating diseases of the central nervous system (1). It is an enveloped virus that contains a positive sense, single-stranded genomic RNA of approximately 31 kilobases in length (2, 3). The viral particle contains four major structural proteins: the nucleocapsid (N) protein that interacts with the viral genomic RNA and membrane (M) protein to form a spherical core (4, 5) and the spike (S) and small membrane (E) proteins that together with the M protein constitute the envelope (6, 7). In some strains of MHV, there exists an additional enveloped protein, the hemagglutinin esterase (8, 9). The E and M proteins were demonstrated to be required for virion assembly (5, 10). The S protein of MHV forms large characteristic projections of coronaviruses and plays a major role in viral pathogenesis.The S protein is cotranslationally glycosylated in the endoplasmic reticulum, giving a molecular mass of approximately 180 kDa, and trimerized (11). Following a transport through the Golgi apparatus, the high mannose oligosaccharides are trimmed, and the protein is further acylated (12). After the modifications, the S protein is often cleaved by a cellular protease into two noncovalently bound 90-kDa subunits, S1 and S2, derived from the N-terminal and C-terminal halves, respectively (13,14). S protein that is not assembled into virions is transported by the secretory system to the cell surface, where it may participate in inducing fusion between adjacent cells (15). In addition to the induction of pH independent cell-to-cell fusion, the S protein also mediates the viral attachment to the receptor of susceptible cells and is involved in the elicitation of neutralizing antibodies, the tissue tropism, and the variation of viral pathogenicity (7, 16 -21).The process of S protein-mediated membrane fusion has been intensively studied, but the mechanism is still poorly understood. Proteolytic cleavage of the S protein precursor into the S1 and S2 subunits was thought to be a prerequisite for the virus to induce cell fusion (14); cell fusion activity correlated with the cleavage level of S protein. The cleavage site was located at the C terminus of the amino acid sequence RRARR in the middle of the S protein (22). Nevertheless, muta...

show abstract

Section: Discussionmentioning

confidence: 99%

A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

Tsai

Chang

1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In order to identify the regions of the S protein structure responsible for its biological functions, it is necessary to express the functional S protein in the absence of other virus-related proteins. Thus, we have previously expressed the entire S protein in cultured mouse cells, using recombinant VV, and showed that the expressed S protein had antigenic and fusogenic properties indistinguishable from those of the authentic S protein (Taguchi et al, 1992;, although the S protein expressed by baculovirus in our laboratory was shown to be similar but not identical to the authentic S protein (Yoden et al, 1989). To study further the regions on the S protein involved in the biological functions, we have expressed the S 1 and $2 subunits of the S protein in a system in which they showed no apparent difference in antigenicity and intracellular transport compared with the authentic S proteins.…”

Section: Discussionmentioning

confidence: 99%

“…All of these plasmids were confirmed to be correctly constructed for the expected S protein-coding regions by dideoxynucleofide chain termination sequencing (Sanger et al, 1977). For expression of the entire S protein of cl-2, we have used the vaccinia virus transfer vector (VV-TV), pSFcl2-S whose construction has been previously described (Taguchi et al, 1992).…”

Section: Methodsmentioning

confidence: 99%

“…In order to express the S1 and $2 proteins, we used cDNA to the S gene of MHV variant cl-2 (Taguchi et al, 1992) shown in Fig. 1 (a).…”

Section: Methodsmentioning

confidence: 99%

“…Murine coronavirus JHM variant cl-2 (Taguchi et al, 1985) and vaccinia virus (VV) strain WR were propagated and assayed using DBT cells and RK13 cells, respectively, as previously described (Taguchi et al, 1992). The growth and maintenance of these cell lines were described elsewhere (Taguchi et al, 1992).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Expression of the S1 and S2 subunits of murine coronavirus JHMV spike protein by a vaccinia virus transient expression system

Kubo

Taguchi

1993

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

The spike (S) protein of murine coronavirus JHMV, variant cl-2, comprises two polypeptides, N-terminal S 1 (with an N-terminal signal peptide) and C-terminal $2 (with a C-terminal transmembrane domain). In order to express these subunits, we constructed three different vaccinia virus transfer vectors (VV-TVs) containing cDNAs encoding the S1 protein without a transmembrane domain (pSFSlutt), the S1 protein with a Cterminal transmembrane domain derived from $2 (pSFSltmd) or the $2 protein with an N-terminal signal peptide derived from S1 (pSFssS2). The S1 and $2 proteins were expressed in DBT cells by infection with vaccinia virus and transfection of these W-TVs. In cells transfected with the pSFSlutt and pSFSltmd, 96K and 106K proteins, respectively, were detected by Western blotting. The ssS2 protein expressed by pSFssS2 was 96K, which was slightly larger than the authentic $2 protein. The Slutt and Sltmd proteins were shown by binding studies using a panel of monoclonal antibodies to be antigenically indistinguishable from the authentic S1 protein. The Sltmd and ssS2 proteins were detected on the cell surface by immunofluorescence, whereas the Slutt protein was not. However, when the Slutt protein was expressed together with the ssS2 protein, the Slutt was detected on the cell membrane. This suggested that the Slutt was associated with ssS2 on the cell membrane. These observations indicate that the expressed S1 and $2 proteins associated in a similar manner to the authentic S1 and $2 proteins produced in DBT cells infected with cl-2. However, cell fusion was not observed in cells expressing either S1 or $2 nor in cells coexpressing both S1 and $2, although the whole S protein expressed by VV-TV did induce fusion.

show abstract

Spike Glycoprotein-Mediated Fusion in Biliary Glycoprotein-Independent Cell-Associated Spread of Mouse Hepatitis Virus Infection

Nash

Buchmeier

1996

Virology

View full text Add to dashboard Cite

The mouse hepatitis virus (MHV) spike glycoprotein mediates attachment of the virus to the MHV receptor, the murine biliary glycoprotein (BGP) carcinoembryonic antigen. Monoclonal antibody CC1 directed against BGP specifically inhibited infection of DBT, Sac-, GT1-7, and OBL21 cells by wild-type MHV-4 and the neuron-adapted variant OBLV60. Binding to this receptor was necessary to establish infection by cell-free MHV; however, the presence of BGP was not required for infection by cell-associated virus. Cell-associated infectious induced syncytium formation on Vero and BHK cells, which lack murine BGP; this activity was not inhibited by monoclonal antibody CC1. Antibody CC1 also did not prevent syncytium formation on DBT cells, which bear BGP. In infectious center assays, the MHV-4 variant OBLV60, which exhibits acid-dependent fusion, spread to cells lacking BGP only when exposed to acidic media. Therefore, spike-mediated fusion was required for BGP-independent spread of MHV infection. Furthermore, BGP-independent, cell-associated spread of MHV-4 was prevented by monoclonal antibodies 5A13.5 and 5B19.2 directed against the spike glycoprotein, but not by other neutralizing and nonneutralizing anti-spike antibodies. Expression of spike glycoprotein by recombinant vaccinia virus resulted in fusion of BGP-negative cells; monoclonal antibodies 5A13.5 and 5B19.2 strongly inhibited spike-mediated fusion in this assay.

show abstract

Molecular cloning and expression of a spike protein of neurovirulent murine coronavirus JHMV variant c1-2

Cited by 45 publications

References 34 publications

A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

Expression of the S1 and S2 subunits of murine coronavirus JHMV spike protein by a vaccinia virus transient expression system

Spike Glycoprotein-Mediated Fusion in Biliary Glycoprotein-Independent Cell-Associated Spread of Mouse Hepatitis Virus Infection

Contact Info

Product

Resources

About