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Identification, sequencing and expression of the glycoprotein L gene of murine cytomegalovirus
1Department of Microbiology, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands.
Abstract:
DNA sequence analysis of the genome of the Smith strain of murine cytomegalovirus (MCMV) revealed an open reading frame (ORF) with amino acid sequence identity to glycoprotein L (gL) of other herpesviruses. The ORF is 822 bp in size and has the capacity to encode a protein of 274 amino acids. It has significant identity with the gL genes of human CMV and human herpesvirus 6. The coding sequence of the gL gene of MCMV strain K181 was also determined, and expressed in Escherichia coli as a fusion protein with glutathione S-transferase using the pGEX expression system. Two antibody-binding regions were identified on the basis of the reactivity of a series of truncated gL constructs with anti-MCMV antibodies. One was mapped to residues 1 to 38 and the other between residues 230 and 274. Polyclonal antibodies specific to gL were raised against the full-length gL fusion protein. The antisera were shown to react with a 46K protein present in purified virions by Western blotting. Treatment of purified virions with endoglycosidase-H or -F resulted in reductions in M(r) of the 46K species to 42K and 31K, respectively. The antisera did not exhibit any neutralizing activity in a plaque reduction assay.
Insights
Researchers identified a gene encoding glycoprotein L (gL) in murine cytomegalovirus (MCMV). Antibodies against this protein did not neutralize MCMV infectivity in vitro.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Murine cytomegalovirus (MCMV) is a model herpesvirus for studying human cytomegalovirus (hCMV).
- Glycoprotein L (gL) is a conserved herpesvirus protein essential for viral entry and assembly.
Purpose of the Study:
- To identify and characterize the gL gene and protein in MCMV.
- To investigate the antigenic properties and potential neutralizing activity of MCMV gL.
Main Methods:
- DNA sequencing of the MCMV genome to identify the gL open reading frame (ORF).
- Expression of MCMV gL in E. coli and generation of polyclonal antibodies.
- Western blotting and endoglycosidase treatment to analyze gL in purified virions.
- Plaque reduction assays to assess antibody-mediated neutralization.
Main Results:
- An 822 bp ORF encoding a 274-amino acid protein with significant homology to gL of other herpesviruses was identified.
- Two antibody-binding regions within MCMV gL were mapped to residues 1-38 and 230-274.
- Polyclonal antibodies raised against MCMV gL recognized a 46 kDa protein in purified virions, which was post-translationally modified.
- These antibodies did not demonstrate neutralizing activity against MCMV in plaque reduction assays.
Conclusions:
- The identified ORF represents the MCMV gL gene.
- MCMV gL is a virion component recognized by specific antibodies.
- MCMV gL, despite its homology to other gL proteins, does not appear to be a target for neutralizing antibodies in this assay.