Bacterial Synthesis of Herpes Simplex Virus Types 1 and 2 Glycoprotein D Antigens

Watson, Roger J.; Weis, John H.; Salstrom, John S.; Enquist, Lynn W.

doi:10.1038/jid.1984.30

Cited by 26 publications

(24 citation statements)

References 20 publications

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“…The 490 serum used in this paper is an example of such a reagent. We have found that such proteins are good immunogens for the preparation of monospecific, polyvalent sera (24,25,36; this report). Since these bacterially produced fusion proteins are generally denatured when injected into animals, the antibodies produced often react with not only authentic native viral proteins, but also with denatured viral protein precursors and products in Western blot analyses (25,36).…”

Section: Discussionmentioning

confidence: 59%

“…A 3-ml sample from each induced culture was harvested at 4 h postinduction. The Cro-PRVgIII fusion proteins were found primarily in insoluble aggregates and were prepared as described previously (36). The aggregated proteins were fractionated by electrophoresis on a 10% SDS-polyacrylamide gel and then stained with Coomassie blue.…”

Section: Methodsmentioning

confidence: 99%

“…coli. The techniques used for producing and purifying Cro-PRV fusion proteins expressed in E. coli have been described previously (24,36).…”

Section: Methodsmentioning

confidence: 99%

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Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture

Robbins¹,

Whealy²,

Watson³

et al. 1986

J Virol

101

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We have established that in the Becker strain of pseudorabies virus (PRV), the glycoprotein glll gene is not essential for growth in cell culture. This was accomplished by construction and analysis of viral mutants containing two defined deletion mutations affecting the glll gene. These mutations were first constructed in vitro and introduced into Escherichia coli expression plasmids to verify structure and protein production. Each mutation was then crossed onto PRV by cotransfection of plasmid DNA and parental viral DNA by using glll-specific monoclonal antibodies as selective and screening reagents. One resultant virus strain, PRV-2, contained an in-frame deletion of a 402-base-pair (bp) Sacl fragment contained within the glll gene. Another virus strain, PRV-10, contained a deletion of a 1,480-bp XhoI fragment removing 230 bp of the upstream, putative transcriptional control sequences and 87% of the gIll coding sequence. The deletion mutants were compared with parental virus by analysis of virion DNA, glll specific RNA, and proteins reacting with glll specific antibodies. Upon infection of PK15 cells, the deletion mutants did not produce any proteins that reacted with two glll specific monoclonal antibodies. However, two species of truncated glycosylated proteins were observed in PRV-2 infected cells that reacted with antiserum raised against bacterially produced gIll protein. PRV-10 produced no detectable gIll-specific RNA or protein. PRV-10 could be propagated without difficulty in tissue culture. Virus particles lacking gIII were indistinguishable from parental PRV virus particles by analysis of infected-cell thin sections in the electron microscope. We therefore conclude that expression of the gIll gene was not absolutely essential for PRV growth in tissue culture. 635on August 1, 2020 by guest

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Section: Discussionmentioning

confidence: 59%

Section: Methodsmentioning

confidence: 99%

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Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture

Robbins¹,

Whealy²,

Watson³

et al. 1986

J Virol

101

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“…The average number of EBNA-1 molecules per cell was measured in a quantitative immunoblot assay with antibodies generated to a bacterially synthesized fusion protein that contains a portion of EBNA-1. DNA encoding part of EBNA-1 was inserted into the bacterial expression plasmid pJS413 (25). This construction is a fusion with an N-terminal portion from the lambda cro gene and a C-terminal portion from the lacZ gene and includes the amino acids from residues 7 to 37 and 420 to 617 of the BKRF1 open reading frame of the B95-8 strain of EBV (1); these residues do not include internal repeat 3 (the Gly-Gly-Ala repeat region of EBNA-1).…”

mentioning

confidence: 99%

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells

Sternås

Middleton

Sugden

1990

J Virol

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Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-l antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. These is no apparent relationship between amounts of EBNA-1 and viral DNA.

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“…It has been used successfully even when the cloned sequence specifies only 10 amino acids of the target gene product (64). By employing a series of monoclonal antibodies, this analysis can be further extended, to map gene sequences that specify a particular epitope (120,122). Thus, lac fusions can be used to identify any gene if an antibody directed against the gene product is available.…”

Section: Identification Of Functional Protein Domainsmentioning

confidence: 99%

Uses of lac fusions for the study of biological problems.

Silhavy¹,

Beckwith²

1985

Microbiological Reviews

192

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Bacterial Synthesis of Herpes Simplex Virus Types 1 and 2 Glycoprotein D Antigens

Cited by 26 publications

References 20 publications

Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture

Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells

Uses of lac fusions for the study of biological problems.

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