2 research outputs found
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