Characterization of gammaherpesvirus 68 gene 50 transcription

Gene 50 is the only immediate-early gene that appears to be conserved among the characterized gammaherpesviruses. It has recently been demonstrated fo r the human viruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associate d herpesvirus (KSHV) that ectopic expression of the gene 50-encoded product in some latently infected cell lines can lead to the induction of virus re plication, indicating that gene 50 is likely to play a pivotal role in regu lating gammaherpesvirus reactivation. Here we demonstrate that the murine g ammaherpesvirus 68 (gamma HV68) gene 50 is an immediate-early gene and that transcription of gamma HV68 gene 50 leads to the production of both splice d and unspliced forms of the gene 50 transcript. Splicing of the transcript near the 5' end serves to extend the gene 50 open reading frame, as has be en observed for the gene 50 transcripts encoded by KSHV and herpesvirus sai miri (Whitehouse ct al., J. Virol. 71:2550-2554, 1997; Lukac et al., Virolo gy 252:304-312, 1998; Sun et al., Proc. Natl. Acad. Sci. USA 95:10866-10871 , 1998). Reverse transcription-PCR analyses, coupled with S1 nuclease prote ction assays, provided evidence that gene 50 transcripts initiate at severa l sites within the region from bp 66468 to 66502 in the gamma HV68 genome. Functional characterization of the region upstream of the putative gene 50 transcription initiation site demonstrated orientation-dependent promoter a ctivity and identified a 110 bp region (bp 66442 to 66552) encoding the put ative gene 50 promoter. Finally, we demonstrate that the gamma HV68: gene 5 0 can transactivate the gamma HV68 gene 57 promoter, a known early gene tar get of the gene 50-encoded transactivator in other gammaherpesviruses. Thes e studies show that the gamma HV68 gene 50 shares several important molecul ar similarities with the gene 50 homologs in other gammaherpesviruses and t hus provides an impetus for future studies analyzing the role of the gamma HV68 gene 50-encoded protein in acute virus replication and reactivation fr om latency in vivo.