The role of specific MHV-68 genes in persistent infection in the lung and virus pathogenesis

Abstract

The gammaherpesvirus subfamily has long been the study of intensive investigation owing to the association between infection and development of lymphoproliferative disease. Well-known members ofthe Gammaherpesvirinae include Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV). Common properties of gammaherpesviruses include a narrow host range of infection and limited productive growth in vitro, and these factors make the study of acute infection problematic. Murine gammaherpesvirus-68 (MHV-68) is able to undergo lytic replication in a range of cell types in vitro and can infect inbred strains of mice. These properties make MHV-68 an excellent model for the study of gammaherpesvirus pathogenesis.Herpesviruses have been indicated in development of diseases in the lung, including pneumonia and idiopathic pulmonary fibrosis. MHV-68 allows investigation of gammaherpesvirus infection of and persistence in the lung - following intranasal inoculation the virus establishes a life-long infection in this organ, with virus persisting in epithelial cells and/or B cells. Identification of key viral genes required for persistence may allow for development of vaccination and/or treatment strategies. Using real-time PCR the long-term viral load in the lungs was reduced following the deletion of key genes from the viral genome. Genes identified are the thymidine kinase gene, previously shown to play a role during acute infection of the lung and ORF73, a homologue of the KSHV LANA-1 gene. Initial data also suggests that the ORF72 and Mil genes, both involved in reactivation from latency, may play a role in maintaining viral load at late time points post-infection.In vivo investigation of the Ml gene of MHV-68 has demonstrated a potential role in control of viral reactivation from latency in the spleen. A novel MHV-68 mutant, MIA, lacking 1171 bp of the Ml ORF, was used to study the role of Ml in pathogenesis. Initial data suggests that in vivo infection with MIA results in increased viral titres during acute infection of the lung, indicating a potential role in control of initial infection. The major role of Ml appears to be during acute phase latency in the spleen, with the MIA virus failing to drive splenomegaly and establishing latency at lower levels. Despite the presence of fewer latently infected splenocytes, MIA reactivates at significantly higher levels, indicating that a function of Ml is to control viral reactivation from latency.A viral mutant (M4Inl) was created that carries a stop codon inserted at genome co¬ ordinate 8386 in the region between the M3 and M4 genes. The mutation is thought to be in an untranscribed region of the genome, potentially in the promoter region of the M3 or M4 genes. Studies demonstrated that the virus is attenuated following infection of both wild-type and IFNyR " mice with respect to lung pathology scores. The lethality of M4Inl in juvenile IFNyR." mice is reduced compared with wild-type MHV-68 infection. Despite the location of the mutation within potential promoter regions, M4Inl transcribes both M3 and M4 at wild type levels in vitro, and in vivo in the spleen. This evidence suggests an apparently untranscribed region of the MHV-68 genome is able to influence pathogenesis in the lung independent of the neighbouring genes.