vOX2- a potential immune regulatory protein involved in Kaposi's sarcoma-associated herpesvirus pathogenesis

Abstract

Kaposi's sarcoma -associated herpesvirus (KSHV), the newest member of the human herpesviruses, was first identified in 1994, and has been shown to be the etiological agent of Kaposi's Sarcoma (KS); a tumour most commonly associated with the onset of AIDS. KSHV is also involved in the development of other malignancies, including Multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL), both of which are B -cell lymphoproliferative disorders. The study of KSHV, as is the case with other herpesviruses from the Gammaherpesvirinae subfamily, is severely limited due to their species specificity and restricted growth in vitro. This has led to the increasing use of closely related murine gammaherpesviruses as a model system to study gammaherpesvirus pathogenesis.A common feature of the gammaherpesviruses is their incorporation of cellular homologous genes during viral evolution, presumably aiding in viral replication and dissemination. One such gene found in KSHV is vOX2, which has homology with the human OX2 gene (hOX2, also recently termed CD200). There are also homologous genes found in mice (mCD200) and rats (rCD200). This membrane bound protein is expressed on a wide variety of cell types, excluding myeloid derived cells. Recent studies have indicated that CD200 plays an important regulatory role in macrophage and microglia activation, through its interaction with a receptor (CD200R) found to be expressed predominantly on macrophage and microglia.Studies on the viral gene, vOX2, have indicated that this glycoprotein has a similar structure to the human CD200, an immunoglobulin superfamily gene, containing two extracellular domains, a single transmembrane region and a short cytoplasmic tail. In order to investigate the potential role of vOX2 in an in vivo system, a novel murid herpesvirus, murine gammaherpesvirus -76 (MHV -76), was utilised. MHV -76 is a natural deletion mutant of murine gammaherpesvirus -68 (MHV -68), lacking four unique genes and eight viral tRNA -like genes from the left end of the MHV -68 genome. This deletion has provided an opportunity to generate a recombinant MHV -76 virus expressing vOX2 (MHV76 -vOX2) and a selectable marker (EGFP -HygR), thus allowing a functional study of vOX2 through the infection of mice. A control recombinant virus (MHV76 -IRES) was also generated, which only expressed the selectable marker (EGFP -HygR). The genomic structures of all recombinant viruses were verified by Southern blot analysis and the expression of the inserted genes was confirmed by northern blot analysis. The growth kinetics of MHV76 -vOX2 and MHV76 -IRES were compared with wild type MHV -76 in both an in vitro single -step and multi -step growth assay. The growth kinetics of the recombinant viruses was not significantly different from that of MHV -76.In vivo studies have indicated that vOX2 has an effect on viral replication; a 10 to 100 -fold increase in viral lung titres was observed in mice infected with MHV76 -vOX2 compared to the control recombinant virus. Examination of lung sections showed that mice infected with the vOX2 recombinant virus elicited a strong influx of inflammatory cells, particularly peripheral mononucleocytes (PMN) and macrophages, resulting in perivascular and peribronchiolar inflammation. Unlike MHV -76, none of the recombinant viruses were able to establish latency in the spleen. Findings from infection of CD200 knock -out mice (C57BL /6 CD2004") with MHV76 -vOX2 support the possibility that vOX2 is playing an important immuno -regulatory role. These studies provide a starting point for further investigation of vOX2 and its potentially important role in KSHV pathogenesis.