Gammaherpesviruses are lymphotropic viruses that establish lifelong latent
infections in their host. The most intensively studied gammaherpesviruses are the
clinically important Epstein Barr virus (EBV) and Kaposi's sarcoma associated
herpesvirus (KSHV). EBV is associated with infectious mononucleosis, Burkitt's
lymphoma, nasopharyngeal carcinoma and Elodgkin's lymphoma. KSHV is almost
certainly the causative agent of Kaposi's sarcoma, body cavity based lymphoma and
multicentric Castleman's disease. Analysis of EBV and KSHV is limited by the
inherent species specificity of these viruses and their restricted growth in vitro.
Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus found in wild
rodents. It productively infects a range of cells and can be grown to high titres.
MHV-68 also infects laboratory mice, thus making it an excellent small animal
model for studying gammaherpesvirus infection. Herpesvirus genomes contain a
variety of homologues of cellular genes involved in immune regulation and/or cell
growth and proliferation. Cellular homologues encoded by MHV-68 include Bcl-2,
cyclin D and a G protein-coupled receptor (GPCR) that shares greatest amino acid
identity with the mammalian chemokine receptor, CXCR2. GPCRs are a
superfamily of seven-transmembrane signalling molecules, and homologues occur in
several herpesviruses. The KSHV GPCR is a functional, constitutively active
oncoprotein that induces vascular endothelial growth factor.
The aim of this project was to characterise the MHV-68 GPCR (ORF74) and
investigate its role in viral pathogenesis. This was approached in several ways.
Firstly, the transcription pattern of the gene was determined using Northern analysis:
GPCR expression was detected at early and late time-points during lytic infection on
multiple rare transcripts. The size of the transcripts suggested they might be
polycistronic and subsequent RT-PCR analysis demonstrated that the GPCR was coexpressed with v-Bcl-2, both in vitro and possibly in vivo. The subcellular
localisation of the GPCR protein was investigated using an "epitope-tagging"
strategy and immunofluorescence experiments revealed an expression pattern
consistent with localisation to the cell surface. The transforming activity of the
GPCR was examined using conventional tissue culture-based assays. NIH3T3 cells
expressing the GPCR exhibited focus formation and anchorage independent growth
in soft agar. Lastly, a recombinant virus lacking the GPCR was generated by
homologous recombination. However, this recombinant virus could not be purified
from the parental wild type virus. In conclusion, the MHV-68 GPCR is a viral
oncogene that is likely to contribute to the long-term persistence of MHV-68