Functional characterisation of alcelaphine herpesvirus-1 ORF 57

Abstract

Malignant catarrhal fever (MCF) or Snotsiekte, a lymphoproliferative disease of both domestic and wild ruminants is characterised by high fever, profuse nasal discharge, lymphadenopathy, leukopenia and severe inflammation of the conjunctival, oral and nasal mucosae. Alcelaphine herpesvirus 1 (A1HV-1) is one of the gammaherpesvirus responsible for the condition in cattle, sheep, deer and other ruminants. A1HV-1 genome has been well characterised and the complete sequence of the genome is available.The open reading frame (ORF) 57 of A1HV-1 is homologous to genes identified in all classes of herpesviruses. The homologues in HSV-1, EBV, HVS and KSHV act as transactivator proteins. The ORF 57 gene product in those viruses has transregulatory function and can either activate or repress viral gene expression by a posttranscriptional mechanism. It encodes for a nuclear protein and has the ability to bind viral RNA, to shuttle between the nucleus and cytoplasm, and is required for efficient nuclear export of viral transcripts.The aim of this study was to functionally characterise the A1HV-1 ORF 57 gene product. RT-PCR experiments have shown that A1F1V-1 ORF 57 is expressed as an immediate early gene. A1HV-1 ORF 57 gene product is a transactivator protein with regulatory functions. In transient transfection assays A1HV-1 ORF 57 gene product has been shown to have effect only on A1HV-1 ORF 50 promoter. The ORF57 gene product up-regulated another immediate early protein encoded by ORF 50 to 50 fold when it is fused to unspliced reporter gene and to 6 fold when it is fused to spliced reporter gene. RT-PCR analysis using RNAs extracted from BHK cells transfected with spliced and unspliced 50 promoter reporter constructs with or without addition of ORJF 57 expression construct have established that ORF 57 gene product acts at 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. The Immunofluorescence studies carried out have shown that ORF 57 protein co-localises with the nucleolar phosphoprotein C23. It was also observed that EGFP-ORF 57 shuttle from the nucleus to the cytoplasm in the presence of Actinomycin D. The functional domain responsible for the nuclear localisation of ORF 57 protein was found to reside at the N-terminus.