Assessment of antigenicity and genetic variation of glycoprotein B of murine cytomegalovirus

Abstract

An analysis of linear antibody-binding sites of the glycoprotein B (gB) molecule ofmurine cytomegalovirus (MCMV) and of genetic variation within these regions was performed. To achieve this, a series of overlapping fragments spanning the entire coding sequence of the gB gene of the K181 strain of MCMV was expressed in E. coli as fusion proteins with glutathione S-transferase (GST) using the pGEX expression system. Four antibody-binding regions were mapped to locations spanning amino acid residues 17-79 (BS), 155-278 (BE2), 809-926 (SS) and 347-508 (BB and EE), based on reactivity in Western blot analysis of GST-gB fusion proteins with murine polyclonal antiserum raised against MCMV. Only the antibody-binding region BE2 (155-278) elicited an antiserum that exhibited complementdependent neutralizing activity, and immunization of mice with the fusion protein BE2 led to moderate but significant reductions in the level of MCMV replication in the spleen. Polyclonal antisera raised against the GST-gB fusion proteins detected purified virion proteins of 105 kDa (anti-BS and anti-BE2) and 52 kDa (anti-SS), and are therefore likely to recognize the N-terminal and C-terminal portions of the gB molecule, respectively. The antibody-binding region within amino acid residues 17-79 was found to be MCMV strain-specific, whereas antibody-binding regions within residues 155-278 and 809-926 were found to be conserved among MCMV field isolates. Comparative sequence analysis of the corresponding regions of MCMV gB revealed a level and extent of sequence heterogeneity consistent with these findings.

Systemic Priming-Boosting Immunization with a Trivalent Plasmid DNA and Inactivated Murine Cytomegalovirus (MCMV) Vaccine Provides Long-Term Protection against Viral Replication following Systemic or Mucosal MCMV Challenge

“…5B and data not shown) that appears on the surface of the mature virion (37). It has been reported that this species may contain at least one neutralizing-antibody epitope as assayed with sera from mice immunized with bacterially expressed gB fragments (63).…”

Functional Consequences of Natural Sequence Variation of Murine Cytomegalovirus m157 for Ly49 Receptor Specificity and NK Cell Activation

“…Secondary SGV stocks were prepared by infecting 3-wk-old female BALB/c mice with 10 3 PFU primary SGV stock. Viral DNA was produced from infected MEFs as previously described (33).…”

Effect of natural sequence variation at the H-2Ld-restricted CD8+ T cell epitope of the murine cytomegalovirus ie1-encoded pp89 on T cell recognition

“…This raises the important consideration of the extent of variation in immunologically important proteins. Recently, we have established that significant genetic variation exists in several regions of the MCMV glycoprotein B molecule, a viral protein that elicits an antiviral antibody response (Xu et al, 1996).…”