Analysis of the in vivo role of the M4 gene of murine gammaherpesvirus-68
Townsley, Alan Craig
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The clinically relevant gammaherpesviruses, Epstein -Barr virus (EBV) and Kaposi's Sarcoma- associated herpesvirus (KSHV), are the subject of intensive research as both are associated with fatal neoplastic disorders. Due to the species specificity and inefficient replication in vitro of EBV and KSHV, several animal models have been proposed to allow analysis of viral pathogenicity in vivo. Murine gammaherpesvirus -68 (MHV -68) represents a tractable animal model of gammaherpesvirus pathogenesis as it is able to infect numerous murid rodent species, including laboratory mice, and replicate productively in a wide range of cell types in vitro, permitting efficient manipulation of the viral genome. MHV -76, a related gammaherpesvirus, is a deletion- mutant of MHV - 68 and lacks 4 MHV -68- specific genes (M1 -M4) and 8 viral tRNA -like sequences at the 5' -end of the genome. These genes are implicated in latency and /or immune evasion. Consequently, MHV -76 is attenuated during productive infection and the early stages of splenic latency, with respect to MHV -68. The aim of the project was the characterisation of the M4 gene of MHV -68. There is little data describing the properties of the M4 gene product; sequence analysis predicted an open- reading frame of 1376óp in length, encoding a -45kDa product. Thus far, M4 expression has been detected during lytic infection, but not during latent infection. Recently, it has been demonstrated that M4 is expressed as an immediate -early /early transcript during lytic replication of MHV -68 in vitro.To elucidate the contribution M4 makes to in vivo pathogenesis, a novel MHV -76 mutant (MHV- 76inM4), in which the region of MHV -68 coding for M4 and accompanying putative promoter elements was inserted into the 5'- region of the MHV - 76 genome, was created. A revertant virus was subsequently generated (MHV- 76.Rev) which restored the 5'- region of the MHV- 76inM4 genome to that of MHV -76. Genomic rearrangements were confirmed by Southern analysis and sequencing. The growth of iv MHV- 76inM4 in vitro was indistinguishable from that of MHV -76 and MHV -68. However, viral titres from MHV- 76inM4- infected BALB /c mice were significantly increased with respect to MHV -76 at early times in the lung, suggesting an important role for M4 during productive infection. Additionally, at days 17 and 21 post- infection, there was a significant elevation in latent viral load in splenocytes of MHV- 76inM4- infected mice compared to MHV -76, as measured by ex vivo reactivation assay and real - time PCR. Like MHV -76, MHV- 76inM4 displays no evidence of overt splenomegaly, characteristic of MHV -68 infection at this time. M4 expression in vivo was detectable by RT -PCR during productive infection in the lung and during the establishment of latency in the spleen, but in general was not detectable during long -term latency. The data demonstrate that M4 modulates both productive and latent infection, and suggest that M4 has a role in subversion of the innate immune response.