Laboratory for Clinical and Molecular Virology,
University of Edinburgh, Scotland
Gammaherpesviruses cause widespread infection in their natural host and are linked to a variety of diseases in man and domestic animals. A feature of all gammaherpesviruses is the ability to infect and persist as a latent infection in lymphocytes and a propensity to produce lymphoproliferative disorders. Although different members of the gammaherpesvirus family are known to target B cells as the principal site of latent infection, it is now clear that other sites of virus persistence/latency exist that may have implications for disease pathogenesis.
Knowledge of gammaherpesvirus latency and cell transformation is largely derived from studies on Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV) and Herpesvirus Saimiri (HVS). In contrast, there is relatively little knowledge of the events preceding the establishment of latency or the diversity of latent infection in vivo with respect to the target cells and the nature of virus gene expression. To address these issues we have been studying murine gammaherpesvirus-68 (MHV-68), a natural pathogen of small rodents (for reviews see Nash et al, 2001; Simas and Efstathiou, 1998).
Intranasal infection of inbred mice with MHV-68 results in virus replication in lung epithelial cells and the establishment of a latent infection in B-lymphocytes, macrophages/dendritic cells and lung epithelial cells. A feature of the primary infection is lymph adenopathy and splenomegaly seen around two weeks post infection that is mediated by virus as well as a CD4+ T cell-driven expansion of B-lymphocytes. At approximately three weeks post infection, an infectious mononucleosis-like syndrome is observed involving an expansion of Vβ4+ CD8+ T cells. At late times post infection, approximately 10% of infected mice develop lymphoproliferative disease (LPD) characterised as lymphomas of B cell origin, with sporadic virus genome positive cells detected within the tumour.
B cells also play a role in trafficking virus around the body since latent infections fail to occur in the spleen of B cell-deficient (µMT) mice. Latent virus is detected in macrophages and dendritic cells in the mediastinal lymph node (MLN) draining the lungs of µMT mice. However, in the absence of B cells the infection in the MLN is not sustained. These findings question the role of macrophages and dendritic cells in maintaining a latent infection in the absence of B cells and suggest that mechanisms of latent infection, in terms of viral gene expression, may be very different between these cell types.
The MHV-68 genome contains approximately 73 open reading frames (ORFs). The majority of the ORFs are co-linear and homologous to other gammaherpesviruses. There are several homologues of cellular genes, such as viral (v)-bcl-2 (M11), G- protein coupled receptor v-GPCR (ORF74), v-cyclin D (ORF72) and a complement regulatory protein (ORF4). To date, a bcl-2 homologue has been shown to be associated with all members of gammaherpesviruses so far studied. MHV-68 also encodes a number of genes that are unique to the virus. These include the genes M1-M4 and eight genes encoding transfer RNA-like structures (v-tRNAs), referred to as the M gene cluster. M1, M3 and M4 gene products are secreted from infected cells, and M3 is a chemokine binding protein. The M gene cluster has been shown to be associated with virus latency; in particular, M2 appears to be transiently expressed in latently infected B cells.
Preliminary studies on lungs from long term infected mice identified the expression of latency-associated genes located at the right end of the genome encoding v-bcl 2, v-cyclin D, v-GPCR and ORF 73 (LANA homologue) (referred to as the Bcl-2 gene cluster) in the absence of expression of lytic cycle genes (e.g. ORF 50). Interestingly, the Bcl-2 gene cluster was not expressed at late times in the spleen, indicating that different states of latent gene expression are associated with this virus – host relationship, similar to that seen for other gammaherpesviruses, such as EBV. Other cellular sites of virus latency and persistence exist which may impact on the overall picture of virus persistence in the lung. For example, alveolar macrophages may play an important role in establishing and maintaining virus persistence, as macrophages have been identified as a site of latent MHV-68 infection. Our hypothesis is that genes in this cluster may have been acquired to promote latency/persistence in sites other than in lymphocytes. Latency is found in the lungs from µMT mice that lack mature B cells.
Other genes associated with latency are located to the M gene cluster. We have characterised another murine gammaherpesvirus, MHV-76. This virus lacks the genes M1 to M4 and all v-tRNA’s, but is otherwise identical to MHV-68. Infection of mice with MHV-76 results in an accelerated clearance from the lung, a dramatic reduction in the incidence of latent infection of lymphocytes and an absence of splenomegaly. One reason for the increased clearance of virus from the lung is the rapid onset of inflammation in MHV-76 compared with MHV-68. We have constructed MHV-68/MHV-76 chimeric viruses in which the deletion in MHV-76 is repaired. This produces a virus identical in biological properties to MHV-68. Using the chimeric virus approach we have started to investigate the function of the M gene cluster and in particular the role of the chemokine binding protein - M3 in interfering with the development of the inflammatory response.
Using a M3- MHV-76 recombinant we demonstrated a delayed inflammatory response in the lung when compared with MHV-76. Similar observations have been made using a MHV-68-M3 deletion virus in which an accelerated inflammatory response was observed, not unlike that seen with MHV-76. M3 has also been implicated in the establishment of latency. This was demonstrated in mice infected with a M3-deleted MHV-68 recombinant which produced a reduced latency phenotype in normal mice but a normal level of latently infected cells in mice deficient in CD8 T cells. This argues in favour of M3 interfering with CD8 T cell recognition of latently infected cells. M3 is structurally a fascinating molecule with the ability to bind to C, CC, CXC, CX3C chemokine families. This is in part due to the dimeric nature of the molecule which exhibits conformational flexibility and electrostatic complementation enabling high-affinity binding of a broad spectrum of chemokines.
The immune response to MHV-68 is characterised by a strong and rapid anti-viral CD8 T cell presence in the lung and lymph nodes of infected mice. These cells detect lytic cycle antigens and are responsible for recovery from the primary infection. CD8 T cells also recognise the latency-associated protein M2. These M2-reactive T-cells may play a role in regulating the level of latent infection in the spleen and in surveillance against reactivating virus. In contrast, the appearance of the neutralizing antibody response to MHV-68 is delayed until the third week post infection. The observation that MHV-68 infects B cells suggests that the virus may have some effect on the evolution of anti-viral antibodies by directly modulating B-cell function.
MHV-68 has been associated with some unusual pathology in mice deficient in IFN-gamma receptors. Such mice develop an overt fibrosis in the draining lymph nodes and spleen which results in a dramatic reduction in lymphocytes between weeks two and three post infection. Intriguingly by week 5 the fibrosed tissues start to recover. Other features of this pathology include: dramatically elevated latency seen in germinal centres, fibrosis of liver and lung also occurs, the fibrosis is reversed by depletion of CD8 T cells and/or CD4 T cells indicating an immune mediated pathology underpinning the fibrosis, loss of splenic architecture. We have also noted other pathology in these mice, notably an arteritis of large blood vessels with virus associated in the media of the vessel walls, boney metaplasia with large numbers of chondrocytes present in affected spleen and a condition similar to primary sclerosing cholangitis, a major fibrosis associated with the bile ducts and liver. We are working on the cellular and molecular characterization of these pathological disorders and their association with virus infection.
From this short review of the features of the biology of MHV-68 infection, it is hoped that parallels can be drawn with Marek’s Disease virus infection and associated disease.
References
Nash, AA, Dutia BM, et al 2001, “Natural history of murine gamma-herpesvirus infection” Philos Trans R Soc Lond B Biol Sci 356: 569-79.
Simas, JP and Efstathiou, S, 1998, “Murine gammaherpesvirus 68: a model for the study of gammaherpesvirus pathogenesis” Trends Microbiol, 6:276-82.
From Proceedings of the “7th International Symposium on Marek’s disease”, St. Catherine's College, Oxford, United Kingdom.
