Transcript analysis of the equine herpesvirus 1 glycoprotein B gene homologue and its expression by a recombinant vaccinia virus.
Abstract: Transcript mapping of the equine herpesvirus 1 (EHV-1) glycoprotein B (gB) gene homologue by Northern blot, S1 nuclease and primer extension analyses indicated that two overlapping transcripts of 3.4 and 4.6 kb originated from the same strand and were transcribed from left to right between coordinates 0.40 and 0.43 of the EHV-1 genome. The 3.4 kb transcript encoded EHV-1 gB and the 5' RNA terminus was located approximately 30 bases downstream from a probable TATA element. The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis and inserted into vaccinia virus by homologous recombination. Cells infected with the recombinant virus synthesized EHV-1 gB antigen, which was detectable in the cytoplasm and on the cell surface by immunofluorescence using an EHV-1 neutralizing horse serum and EHV-1 monoclonal antibodies. On Western blots, bands of 138K to 143K, 80K to 90K and 55K to 57K were identified in recombinant virus-infected cells, by both EHV-1 monoclonal antibodies and the polyclonal horse serum. These were similar in Mr to bands identified by these sera in EHV-1-infected cells. Mice vaccinated with the recombinant virus produced antibodies which recognized proteins of the same Mr as EHV-1 gB, on Western blots, but did not have in vitro neutralizing activity.
Publication Date: 1990-05-01 PubMed ID: 2161047DOI: 10.1099/0022-1317-71-5-1119Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article is focused on the transcript mapping of the equine herpesvirus 1 (EHV-1) glycoprotein B (gB) gene and its expression by a recombinant vaccinia virus, with experimental findings that provide informative details on the structure and expression of the EHV-1 gB gene.
Transcript Mapping of EHV-1 gB Gene
- The researchers carried out a transcript mapping of the EHV-1 gB gene homologue using procedures like Northern blot, S1 nuclease and primer extension analyses.
- This process illuminated that two overlapping transcripts of 3.4 and 4.6 kb were originated from the same strand and were transcribed from left to right, occupying the region between coordinates 0.40 and 0.43 of the EHV-1 genome.
- The smaller transcript of 3.4 kb was found to be encoding the EHV-1 gB and its 5′ RNA terminus was located approximately 30 bases downstream to a probable TATA element. This piece of evidence sheds light on the coding context of the gB gene.
Reconstruction and Recombination of gB Gene Homologue
- The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis. Oligonucleotide mutagenesis is a method employed for the amplification and modification of DNA.
- After reconstruction, the gB gene homologue was inserted into the vaccinia virus by a biological process known as homologous recombination.
Effects of Recombinant Vaccinia Virus Infection
- The cells infected with the recombinant virus synthesized the EHV-1 gB antigen. This antigen was detectable both in the cytoplasm and on the cell surface when tested with EHV-1 neutralizing horse serum and EHV-1 monoclonal antibodies using a method named immunofluorescence.
- On Western blots, certain protein bands (from 55K to 143K) specific to the recombinant virus were identified in the cells infected with it. The bands were similar in size (molecular weight-Mr) to bands identified in EHV-1-infected cells.
Mouse Vaccination Using Recombinant Virus
- Mice vaccinated with the recombinant virus produced antibodies that recognized proteins of the same size as the EHV-1 gB on Western blots.
- Despite this recognition, these antibodies did not demonstrate an in vitro neutralizing activity.
Cite This Article
APA
Bell CW, Boyle DB, Whalley JM.
(1990).
Transcript analysis of the equine herpesvirus 1 glycoprotein B gene homologue and its expression by a recombinant vaccinia virus.
J Gen Virol, 71 ( Pt 5), 1119-1129.
https://doi.org/10.1099/0022-1317-71-5-1119 Publication
Researcher Affiliations
- School of Biological Sciences, Macquaire University, Sydney, Australia.
MeSH Terms
- Animals
- Base Sequence
- Blotting, Northern
- Blotting, Western
- Cell Line
- Cloning, Molecular
- Gene Expression Regulation, Viral
- Genes, Viral
- Herpesviridae / genetics
- Herpesvirus 1, Equid / genetics
- Immunization
- Mice
- Mice, Inbred CBA
- Molecular Sequence Data
- Neutralization Tests
- Restriction Mapping
- Sequence Homology, Nucleic Acid
- Transcription, Genetic
- Vaccinia virus / genetics
- Viral Envelope Proteins / genetics
- Viral Envelope Proteins / immunology
Citations
This article has been cited 7 times.- Kim SK, Shakya AK, O'Callaghan DJ. Immunization with Attenuated Equine Herpesvirus 1 Strain KyA Induces Innate Immune Responses That Protect Mice from Lethal Challenge. J Virol 2016 Sep 15;90(18):8090-104.
- Smith PM, Zhang Y, Jennings SR, O'Callaghan DJ. Characterization of the cytolytic T-lymphocyte response to a candidate vaccine strain of equine herpesvirus 1 in CBA mice. J Virol 1998 Jul;72(7):5366-72.
- Wellington JE, Love DN, Whalley JM. Evidence for involvement of equine herpesvirus 1 glycoprotein B in cell-cell fusion. Arch Virol 1996;141(1):167-75.
- Sinclair R, Binns MM, Chirnside ED, Mumford JA. Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B. J Clin Microbiol 1993 Feb;31(2):265-71.
- Bell CW, Whalley JM. Herpesvirus ICP18.5 and DNA-binding protein genes are conserved in equine herpesvirus-1. Virus Genes 1993 Sep;7(3):219-28.
- Whalley M, Robertson G, Bell C, Love D, Elphinstone M, Wiley L, Craven D. Identification and comparative sequence analysis of a gene in equine herpesvirus 1 with homology to the herpes simplex virus glycoprotein D gene. Virus Genes 1991 Oct;5(4):313-25.
- Maeda K, Horimoto T, Norimine J, Kawaguchi Y, Tomonaga K, Niikura M, Kai C, Takahashi E, Mikami T. Identification and nucleotide sequence of a gene in feline herpesvirus type 1 homologous to the herpes simplex virus gene encoding the glycoprotein B. Arch Virol 1992;127(1-4):387-97.
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