Functional characterisation of Alcelaphine Herpesvirus-1 ORF 57

• Main Author: Thonur, Leena Devi
• Published: University of Edinburgh 2002
• Subjects: 636.089
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662932

The aim of this study was to functionally characterise the A1HV-1 ORF 57 gene product. The studies carried out have shown that A1HV-1 ORF 57 is expressed as an immediate early gene. It has also been demonstrated that the ORF 57 gene product is a transactivator protein with post-transcriptional regulatory functions. In transient transfection assays A1HV-1 ORF 57 gene product has been shown to activate transcription from only the A1HV-1 ORF 50 promoter. The ORF57 gene product up-regulated the activity of an unspliced reporter gene driven by ORF 50 promoter to 50 fold and of a spliced reporter gene driven by ORF 50 promoter to 6 fold: RT-PCR analysis using RNAs extracted from BHK cells transfected with spliced and unspliced 50 promoter reporter constructs with or without addition of ORF 57 expression construct have demonstrated that the increase in the amount of protein, produced was not correlated with a similar increase in the mRNA level. This indicated that the ORF 57 gene product acts at a post-transcriptional level. Transient transfection assays with full length ORF 57 and truncations of ORF 57 have demonstrated that the activation domain of ORF 57 lies at the C-terminus. When ORF 57 was fused to the enhanced green fluorescent protein (EGFP), the fusion protein exhibited a punctate nuclear distribution in transfected BHK cells. Immunofluorescence studies have shown that ORF 57 protein co-localises with the nucleolar phosphoprotein C23. It was also observed that EGFP-ORF 57 shuttles from the nucleus to the cytoplasm in the presence of Actinomycin D. The functional domain responsible for the nuclear localisation of ORF57 protein was found to reside at the N-terminus. Thus, this study has demonstrated that A1HV-1 ORF 57 gene product shares some of the functional properties with other gammaherpesvirus homologues.