ABSTRACT
Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.
Subject(s)
Gammaherpesvirinae/chemistry , Membrane Glycoproteins/analysis , Viral Envelope Proteins/analysis , Virion/chemistry , Amino Acid Sequence , Animals , Base Sequence , Gammaherpesvirinae/genetics , Genes, Viral , Mice , Molecular Sequence Data , Molecular Weight , Viral Envelope Proteins/immunologyABSTRACT
The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 is essential for viral genome maintenance in vitro and may be the only EBV protein expressed by the majority of latently infected cells in vivo. EBNA-1 may therefore be critical to the evasion of host immunity which allows persistent infection. EBNA-1 includes a polymorphic internal repeat domain of unknown significance and unique regions which mediate all known functional activities and which have hitherto been assumed to be conserved between strains. Monoclonal antibodies were generated using a construct based on EBNA-1 of the prototype B95-8 strain, deleted for the repeat domain. These antibodies showed a limited profile of recognition of EBNA-1 in common laboratory EBV+ cell lines by immunoprecipitation and immunostaining. The observed antigenic heterogeneity also extended to spontaneously transformed B lymphoblastoid cell lines (LCLs) representing viral isolates circulating within US and UK populations. DNA fragments spanning the C-terminal unique domain of EBNA-1 from eleven spontaneous LCLs were amplified by polymerase chain reaction for sequencing, which directly demonstrated extensive and unexpected variability between diverse type 1 EBV isolates. The resulting polymorphism affects most of the putative MHC Class I binding epitopes which could be identified within this region using published sequence motifs, and influences MHC binding by variants of at least one such peptide in the processing mutant cell line T2. These findings could be related to the apparent lack of recognition of EBNA-1 by cytotoxic T lymphocytes.
Subject(s)
Antigenic Variation/genetics , Antigens, Viral/genetics , DNA-Binding Proteins/genetics , Genetic Variation/genetics , Herpesvirus 4, Human/genetics , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Antigens, Viral/chemistry , Antigens, Viral/immunology , Antigens, Viral/metabolism , B-Lymphocytes/virology , Base Sequence , Cell Line, Transformed , DNA, Viral/analysis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Epitopes/genetics , Epitopes/immunology , Epstein-Barr Virus Nuclear Antigens , HLA-A2 Antigen/metabolism , Herpesvirus 4, Human/immunology , Humans , Molecular Sequence Data , Recombinant Fusion Proteins/immunology , Sequence Analysis, DNA , T-Lymphocytes/virologyABSTRACT
Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of murid rodents and displays similar pathobiological characteristics to those of the human gammaherpesvirus Epstein-Barr virus (EBV). However, in contrast to EBV, MHV-68 will replicate in epithelial cells in vitro. It has therefore been proposed that MHV-68 may be of use as a model for the study of gammaherpesviruses, EBV in particular, both in vitro and in vivo. The EBV homolog of herpes simplex virus glycoprotein B (gB), termed gp110, is somewhat unusual compared with those of many other herpesviruses. We therefore decided to characterize the homolog of gB encoded by MHV-68 (termed MHV gB) to observe the properties of a gammaherpesvirus gB produced in epithelial cells and also to test the relatedness of MHV-68 and EBV. The MHV gB-coding sequence was determined from cloned DNA. The predicted amino acid sequence shared closest homology with gammaherpesvirus gB homologs. Biochemical analysis showed that MHV gB was a glycoprotein with a molecular weight of 105,000. However, the glycans were of the N-linked, high-mannose type, indicating retention in the endoplasmic reticulum. In line with this, MHV gB was localized to the cytoplasm and nuclear margins of infected cells but was not detected on the cell surface or in virions. Additionally, anti-MHV gB antisera were nonneutralizing. Thus, the MHV gB was unlike many other herpesvirus gBs but was extremely similar to the EBV gB. This highlights the close relationship between MHV-68 and EBV and underlines the potential of MHV-68 as a model for EBV in epithelial cells both in vitro and in vivo.
