Abstract
We have established that in the Becker strain of pseudorabies virus (PRV), the glycoprotein gIII 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 gIII 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 gIII-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) SacI fragment contained within the gIII 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 gIII coding sequence. The deletion mutants were compared with parental virus by analysis of virion DNA, gIII specific RNA, and proteins reacting with gIII specific antibodies. Upon infection of PK15 cells, the deletion mutants did not produce any proteins that reacted with two gIII 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 gIII protein. PRV-10 produced no detectable gIII-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 gIII gene was not absolutely essential for PRV growth in tissue culture.
Full text
PDF










Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ben-Porat T., DeMarchi J., Pendrys J., Veach R. A., Kaplan A. S. Proteins specified by the short unique region of the genome of pseudorabies virus play a role in the release of virions from certain cells. J Virol. 1986 Jan;57(1):191–196. doi: 10.1128/jvi.57.1.191-196.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ben-Porat T., Demarchi J. M., Kaplan A. S. Characterization of defective interfering viral particles present in a population of pseudorabies virions. Virology. 1974 Sep;61(1):29–37. doi: 10.1016/0042-6822(74)90239-6. [DOI] [PubMed] [Google Scholar]
- Ben-Porat T., Kaplan A. S. Synthesis of proteins in cells infected with herpesvirus. V. Viral glycoproteins. Virology. 1970 Jun;41(2):265–273. doi: 10.1016/0042-6822(70)90078-4. [DOI] [PubMed] [Google Scholar]
- Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
- Berk A. J., Sharp P. A. Structure of the adenovirus 2 early mRNAs. Cell. 1978 Jul;14(3):695–711. doi: 10.1016/0092-8674(78)90252-0. [DOI] [PubMed] [Google Scholar]
- Bzik D. J., Fox B. A., DeLuca N. A., Person S. Nucleotide sequence specifying the glycoprotein gene, gB, of herpes simplex virus type 1. Virology. 1984 Mar;133(2):301–314. doi: 10.1016/0042-6822(84)90397-0. [DOI] [PubMed] [Google Scholar]
- Cohen G. H., Long D., Eisenberg R. J. Synthesis and processing of glycoproteins gD and gC of herpes simplex virus type 1. J Virol. 1980 Nov;36(2):429–439. doi: 10.1128/jvi.36.2.429-439.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Denniston K. J., Madden M. J., Enquist L. W., Vande Woude G. Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles. Gene. 1981 Dec;15(4):365–378. doi: 10.1016/0378-1119(81)90180-3. [DOI] [PubMed] [Google Scholar]
- Dowbenko D. J., Lasky L. A. Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene. J Virol. 1984 Oct;52(1):154–163. doi: 10.1128/jvi.52.1.154-163.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eisenberg R. J., Long D., Ponce de Leon M., Matthews J. T., Spear P. G., Gibson M. G., Lasky L. A., Berman P., Golub E., Cohen G. H. Localization of epitopes of herpes simplex virus type 1 glycoprotein D. J Virol. 1985 Feb;53(2):634–644. doi: 10.1128/jvi.53.2.634-644.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frink R. J., Eisenberg R., Cohen G., Wagner E. K. Detailed analysis of the portion of the herpes simplex virus type 1 genome encoding glycoprotein C. J Virol. 1983 Feb;45(2):634–647. doi: 10.1128/jvi.45.2.634-647.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Hampl H., Ben-Porat T., Ehrlicher L., Habermehl K. O., Kaplan A. S. Characterization of the envelope proteins of pseudorabies virus. J Virol. 1984 Nov;52(2):583–590. doi: 10.1128/jvi.52.2.583-590.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holland T. C., Homa F. L., Marlin S. D., Levine M., Glorioso J. Herpes simplex virus type 1 glycoprotein C-negative mutants exhibit multiple phenotypes, including secretion of truncated glycoproteins. J Virol. 1984 Nov;52(2):566–574. doi: 10.1128/jvi.52.2.566-574.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holland T. C., Sandri-Goldin R. M., Holland L. E., Marlin S. D., Levine M., Glorioso J. C. Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay. J Virol. 1983 May;46(2):649–652. doi: 10.1128/jvi.46.2.649-652.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mettenleiter T. C., Lukacs N., Rziha H. J. Mapping of the structural gene of pseudorabies virus glycoprotein A and identification of two non-glycosylated precursor polypeptides. J Virol. 1985 Jan;53(1):52–57. doi: 10.1128/jvi.53.1.52-57.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mettenleiter T. C., Lukàcs N., Rziha H. J. Pseudorabies virus avirulent strains fail to express a major glycoprotein. J Virol. 1985 Oct;56(1):307–311. doi: 10.1128/jvi.56.1.307-311.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pancake B. A., Aschman D. P., Schaffer P. A. Genetic and phenotypic analysis of herpes simplex virus type 1 mutants conditionally resistant to immune cytolysis. J Virol. 1983 Sep;47(3):568–585. doi: 10.1128/jvi.47.3.568-585.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pellett P. E., Biggin M. D., Barrell B., Roizman B. Epstein-Barr virus genome may encode a protein showing significant amino acid and predicted secondary structure homology with glycoprotein B of herpes simplex virus 1. J Virol. 1985 Dec;56(3):807–813. doi: 10.1128/jvi.56.3.807-813.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pellett P. E., Kousoulas K. G., Pereira L., Roizman B. Anatomy of the herpes simplex virus 1 strain F glycoprotein B gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibody-resistant mutants. J Virol. 1985 Jan;53(1):243–253. doi: 10.1128/jvi.53.1.243-253.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rea T. J., Timmins J. G., Long G. W., Post L. E. Mapping and sequence of the gene for the pseudorabies virus glycoprotein which accumulates in the medium of infected cells. J Virol. 1985 Apr;54(1):21–29. doi: 10.1128/jvi.54.1.21-29.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
- Robbins A. K., Watson R. J., Whealy M. E., Hays W. W., Enquist L. W. Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C. J Virol. 1986 May;58(2):339–347. doi: 10.1128/jvi.58.2.339-347.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robbins A. K., Weis J. H., Enquist L. W., Watson R. J. Construction of E. coli expression plasmid libraries: localization of a pseudorabies virus glycoprotein gene. J Mol Appl Genet. 1984;2(5):485–496. [PubMed] [Google Scholar]
- Roizman B., Carmichael L. E., Deinhardt F., de-The G., Nahmias A. J., Plowright W., Rapp F., Sheldrick P., Takahashi M., Wolf K. Herpesviridae. Definition, provisional nomenclature, and taxonomy. The Herpesvirus Study Group, the International Committee on Taxonomy of Viruses. Intervirology. 1981;16(4):201–217. doi: 10.1159/000149269. [DOI] [PubMed] [Google Scholar]
- Smith K. O., Kennell W. L., Lamm D. L. Visualization of minute centers of viral infection in unfixed cell cultures by an enzyme-linked antibody assay. J Immunol Methods. 1981;40(3):297–305. doi: 10.1016/0022-1759(81)90361-6. [DOI] [PubMed] [Google Scholar]
- Snowden B. W., Halliburton I. W. Identification of cross-reacting glycoproteins of four herpesviruses by Western blotting. J Gen Virol. 1985 Sep;66(Pt 9):2039–2044. doi: 10.1099/0022-1317-66-9-2039. [DOI] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Swain M. A., Peet R. W., Galloway D. A. Characterization of the gene encoding herpes simplex virus type 2 glycoprotein C and comparison with the type 1 counterpart. J Virol. 1985 Feb;53(2):561–569. doi: 10.1128/jvi.53.2.561-569.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wathen M. W., Wathen L. M. Isolation, characterization, and physical mapping of a pseudorabies virus mutant containing antigenically altered gp50. J Virol. 1984 Jul;51(1):57–62. doi: 10.1128/jvi.51.1.57-62.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watson R. J. DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene. Gene. 1983 Dec;26(2-3):307–312. doi: 10.1016/0378-1119(83)90203-2. [DOI] [PubMed] [Google Scholar]
- Watson R. J., Weis J. H., Salstrom J. S., Enquist L. W. Herpes simplex virus type-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli. Science. 1982 Oct 22;218(4570):381–384. doi: 10.1126/science.6289440. [DOI] [PubMed] [Google Scholar]