Analysis of the Functions of Coronavirus Glycoproteins by Differential Inhibition of Synthesis with Tunicamycin

Holmes, Kathryn V.; Doller, Elizabeth W.; Behnke, James

doi:10.1007/978-1-4757-0456-3_11

Cited by 59 publications

(56 citation statements)

References 9 publications

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“…The results presented here confirm earlier studies (Siddell et al, 1980(Siddell et al, , 1981bAnderson et al, 1979;Rottier et al, 1981b;Bond et al, 1979Bond et al, , 1981Gerdes et al, 1981;Holmes et al, 1981) which show that the major polypeptide synthesized in MHV-infected cells is the intracellular form of the phosphorylated nucleocapsid protein. Fig.…”

Section: Discussionsupporting

confidence: 92%

“…Because it has been shown for MHV-A59 or MHV-JHM that the higher molecular weight form of both the intracellular and virion matrix protein species are glycosylated (Siddell et al, 1981b;Sturman & Holmes, 1977;Neimann & Klenk, 1981;Macnaughton, 1980;, it is logical to conclude that this modification is due to glycosylation. A number of groups have now demonstrated that this glycosylation event is not, however, inhibited by the drug tunicamycin (Niemann & Klenk, 1981;Rottier et al, 1981b;Siddell et aL, 1981b;Holmes et al, 1981;) and chemical analysis conclusively shows that the glycosidic linkage is of the O type (Niemann & Klenk, 1981). Recently, by a combination of tryptic peptide mapping and partial proteolytic analysis, Stern et al (1982) have concluded that the analogous proteins in the infectious bronchitis virion, i.e.…”

Section: Discussionmentioning

confidence: 99%

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Coronavirus JHM: Tryptic Peptide Fingerprinting of Virion Proteins and Intracellular Polypeptides

Siddell¹

1982

Journal of General Virology

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SUMMARYThe virion proteins and intracellular polypeptides of the murine coronavirus MHV-JHM have been analysed by two-dimensional fingerprinting of their [a~S]methionine-containing tryptic peptides. The analysis shows that the virion proteins gp98, gp65, pp60 and p23 are distinct. Virion protein gp25 has the same polypeptide component as p23, and virion protein gpl70 has a polypeptide component related to gp98. The six virus polypeptides synthesized in infected cells, I50K, 65K, 60K, 30K, 23K and 14K are also distinct. The 170K and 98K species, which are produced by processing, are related to 150K. The 25K species is a processed form of 23K. The identity of corresponding species in the cell and in the virion has been shown and a model describing the genesis of coronavirus JHM proteins can now be proposed.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Coronavirus JHM: Tryptic Peptide Fingerprinting of Virion Proteins and Intracellular Polypeptides

Siddell¹

1982

Journal of General Virology

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“…The ef®ciency of fractionation of the cytoplasm into soluble and membrane portions was monitored by following the partition of the MHV N (lanes 1 and 2) and M (lanes 3 and 4) proteins, which are known to be free in the cytoplasm (N) or localized to the intermediate compartment and the Golgi (M) (Holmes et al, 1981;Collins et al, 1982). The p28 protein was precipitated predominantly from the soluble cytoplasmic fraction (lane 5) rather than the membrane pellet (lane 6).…”

Section: Resultsmentioning

confidence: 99%

Localization of mouse hepatitis virus open reading frame 1A derived proteins

Bi¹,

Piñón²,

Hughes³

et al. 1998

J Neurovirol

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We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5' portion of mouse hepatitis virus (MHV) gene 1. Immuno¯uorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewhat granular cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm of MHV-infected, but not control uninfected cells. Immunouorescent staining of infected cells with antisera which recognize the downstream polypeptides, p65, p240 and p290 labeled discrete vesicular perinuclear structures. Double immuno¯uorescent labeling of BHK cells expressing the MHV receptor (BHK MHVR1 ) and infected with MHV-A59 with a Golgi-speci®c anti-mannosidase II monoclonal antibody and with antiserum recognizing each of these anti-MHV ORF1a polypeptides, showed that the p240 and p290 polypeptides were localized in discrete vesicular structures that overlapped the Golgi complex. Labeling with antibodies speci®c for p65 colocalized with the Golgi region, and showed staining of the perinuclear cytoplasm as well. Plasmids containing sequences contained in the ®rst 6.75 kb of ORF1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Immuno¯uorescent labeling of transfectants with the anti-ORF1a antisera showed patterns of antigen distribution similar to those observed in cells infected with MHV-A59. A deletion analysis with constructs containing only portions of the ORF1a sequence indicated that 303 amino acids containing the ®rst papain-like protease domain (PLP-1) was suf®cient to associate this protein with the Golgi.

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“…The spike consists of dimers or trimers of the S protein proteolytically cleaved into two subunits, the N-terminal S1 and the C-terminal $2 (de Groot et al, 1987). The S protein has important biological functions (Holmes et al, 1981;Collins et al, 1982;Sturman et al, 1985); it has been reported that the S protein binds to receptors on the surface of susceptible cells (Collins et al, 1982), and it is also involved in the syncytium formation observed in MHVinfected cells (Collins et al, 1982). This fusogenic property of the S protein has been reported to manifest only after cleavage Frana et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

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

Taguchi¹,

Ikeda²,

Shida³

1992

Journal of General Virology

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A cDNA encoding the spike (S) protein of the neurovirulent murine coronavirus JHMV variant cl-2 was isolated and sequenced. Analysis of the cDNA revealed that the S protein consists of 1376 amino acids, as does the S protein of mouse hepatitis virus 4. We inserted the cDNA into the genome of vaccinia virus to obtain a recombinant vaccinia virus (rVV). The S protein expressed in RK13 cells infected by the rVV was shown to be electrophoretically and immunologically indistinguishable from the S protein produced in DBT cells infected with cl-2 virus. RVV infection of rats and mice induced S protein-specific antibody production detectable by immunofluorescence and neutralization. Moreover, the S protein expressed by the rVV induced syncytium formation not only in mouse DBT and L cells, which are susceptible to cl-2 virus infection, but also in rabbit RK13 cells, which are not susceptible to cl-2 virus infection. This result suggests the possibility that RK13 cells have binding sites for the cl-2 virus S protein.

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Analysis of the Functions of Coronavirus Glycoproteins by Differential Inhibition of Synthesis with Tunicamycin

Cited by 59 publications

References 9 publications

Coronavirus JHM: Tryptic Peptide Fingerprinting of Virion Proteins and Intracellular Polypeptides

Coronavirus JHM: Tryptic Peptide Fingerprinting of Virion Proteins and Intracellular Polypeptides

Localization of mouse hepatitis virus open reading frame 1A derived proteins

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

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