Characterisation of Alcelaphine herpesvirus-1 ORF50

Malignant catarrhal fever (MCF) is a lymphoproliferative, degenerative and often fatal disease of members of the Artiodactyla family such as cattle and deer. The causal agents of MCF are a group of gammaherpesviruses of which alcelaphine herpesvirus-1 (A1HV-1) is a member. A1HV-1 is the most well characterised of the group and its genome has been sequenced. Continued passage of A1HV-1 in bovine cells results in attenuation of the virus. Comparison of genomes from wild-type and attenuated viruses suggested that open reading frames (ORFs) are affected including ORF50.The ORF50 gene products of Kaposi's sarcoma-associated herpesvirus (KSHV), herpesvirus saimiri (HVS), murine gammaherpesvirus-68 (MHV-68) and their equivalent, the BRLF1 gene product of Epstein-Barr virus (EBV) are called Rtransactivators (Rtas). They have a crucial role in the key mechanism of reactivating the virus from latency as well as acting as transactivator proteins activating a variety of virus and cellular promoters.The aim of this study was to characterise A1HV-1 ORF50. It was demonstrated that the ORF50 gene product, referred to as A1HV-1/Rta, acted as a transactivator. The ability to transactivate three A1HV-1 promoters was investigated. It was shown that A1HV-1/Rta activates A1HV-1 ORF57 and A1HV-1 ORF6 putative promoters but not the thymidine kinase putative promoter. The ORF57 promoter was examined and the transcriptional start site and splice acceptor and splice donor sites were located. Also, activation of the ORF57 promoter by A1HV-1/Rta was investigated further.Truncated ORF57 promoters were generated and their ability to be activated by A1HV-1/Rta was investigated. It was found that A1HV-1/Rta required sequences at least 385 bp upstream of the ORF57 transcriptional start site to exert its effect on ORF57 transcription. A potential AlHV-l/Rta-responsive region was identified and this was investigated further using electrophoretic mobility shift assays.A second approach to characterise A1HV-1 ORF50 was also taken. Various strategies were designed to generate a recombinant A1HV-1 lacking ORF50. The method pursued was to generate a bacterial artificial chromosome containing the entire A1HV-1 genome. These strategies will be discussed