Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells

Pachl, Carol; Burke, Rae Lyn; Stuve, Laura L.; Sanchez-Pescador, Lisa; Nest, Gary Van; Masiarz, Frank R.; Dina, Dino

doi:10.1128/jvi.61.2.315-325.1987

Cited by 67 publications

(23 citation statements)

References 60 publications

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“…Although perinuclear accumulation of gI has also been described for BHV-1-infected bovine cells (37), the accumulation observed in the transfected murine cell clones expressing gI appeared to be greater. This observation is similar to those reported for expression of recombinant HSV gB in Chinese hamster ovary and COS cell lines, in which cell-associated or perinuclear localization was a prominent feature of the intracellular distribution of this closely related protein (2,38).…”

Section: Vol 62 1988supporting

confidence: 90%

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

FitzPatrick

Zamb

Parker

et al. 1988

J Virol

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Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIll, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTKcells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for glll, a deficiency In 0-linked glycosylation was implicated. The reactivity pattern of a panel of gI-and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-l-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and glll by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIll as targets of the immune response to BHV-1 in this murine model system.

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Section: Vol 62 1988supporting

confidence: 90%

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

FitzPatrick

Zamb

Parker

et al. 1988

J Virol

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“…An estimate of the copy number was made by comparison of the intensity of the bands obtained with those generated by amplification of known amounts of HSV-2 DNA. The precise copy number was then determined by coamplification of serial twofold dilutions of the DNA with 1,000 copies of linearized plasmid pHS108, which carries the entire glycoprotein B gene (8). Products were detected by liquid hybridization.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of a quantitative competitive PCR assay for measuring herpes simplex virus DNA content in genital tract secretions

et al. 1997

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Previous studies have shown an association between the approximate titer of herpes simplex virus (HSV) DNA in clinical specimens and the ability to isolate HSV from genital secretions. To control for variance in amplification conditions, we developed a competitive quantitative PCR (QC PCR) for the detection of HSV DNA. The assay accurately measured from 10 to 10(6) copies of HSV DNA. We compared the QC PCR with our previous semiquantitative detection method and found concordance for 61 of 63 positive specimens. We also evaluated the HSV DNA content from individual swabs of genital secretions obtained from individual sites of the genital tract (cervix, vulva, and rectum) with that from one swab with secretions from all three sites. The concordance for detecting HSV DNA was 91%; for only 4 of 143 collection days was there a > 1 log difference between the two collection methods. A single swab with secretions from all three genital sites and evaluated in a QC PCR format can accurately measure the frequency of subclinical and clinical shedding of HSV and the titer of HSV shed from the genital region. Such an approach should be very useful in the evaluation of antiviral chemotherapy for HSV.

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“…Detection of antibody to HSV glycoprotein by ELISA (Pachl et al, 1987) was performed as previously described (Meseda et al, 2002;Nass et al, 1998), using a mixture of HSV-2 glycoproteins or an inactivated HSV-2-infected cell extract. Endpoint titer was defined as the highest dilution of serum that gave an A 405 value that was 2-fold greater than that of the matched dilution of normal prebleed mouse serum and was also greater than 0.050.…”

Section: Antibody and Neutralizing Antibody Determinationmentioning

confidence: 99%

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

et al. 2004

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Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.

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Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells

Cited by 67 publications

References 60 publications

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

Evaluation of a quantitative competitive PCR assay for measuring herpes simplex virus DNA content in genital tract secretions

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

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