Alternatively Initiated Gene 50/RTA Transcripts Expressed during Murine and Human Gammaherpesvirus Reactivation from Latency

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In the process of characterizing the requirements for expression of the essential immediate-early transcriptional activator (RTA) encoded by gene 50 of murine gammaherpesvirus 68 (MHV68), a recombinant virus was generated in which the known gene 50 promoter was deleted (G50pKO). Surprisingly, the G50pKO mutant retained the ability to replicate in permissive murine fibroblasts, albeit with slower kinetics than wild-type MHV68. 5'-rapid amplification of cDNA ends analyses of RNA prepared from G50pKO-infected fibroblasts revealed a novel upstream transcription initiation site, which was also utilized during wild-type MHV68 infection of permissive cells. Furthermore, the region upstream of the distal gene 50/RTA transcription initiation site exhibited promoter activity in both permissive NIH 3T12 fibroblasts as well as in the murine macrophage cell line RAW 264.7. In addition, in RAW 264.7 cells the activity of the distal gene 50/RTA promoter was strongly upregulated (>20-fold) by treatment of the cells with lipopolysaccharide. Reverse transcriptase PCR analyses of RNA prepared from Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-infected B-cell lines, following induction of virus reactivation, also revealed the presence of gene 50/RTA transcripts initiating upstream of the known transcription initiation site. The latter argues that alternative initiation of gene 50/RTA transcription is a strategy conserved among murine and human gammaherpesviruses. Infection of mice with the MHV68 G50pKO demonstrated the ability of this mutant virus to establish latency in the spleen and peritoneal exudate cells (PECs). However, the G50pKO mutant was unable to reactivate from latently infected splenocytes and also exhibited a significant reactivation defect from latently infected PECs, arguing in favor of a model where the proximal gene 50/RTA promoter plays a critical role in virus reactivation from latency, particularly from B cells. Finally, analyses of viral genome methylation in the regions upstream of the proximal and distal gene 50/RTA transcription initiation sites revealed that the distal promoter is partially methylated in vivo and heavily methylated in MHV68 latently infected B-cell lines, suggesting that DNA methylation may serve to silence the activity of this promoter during virus latency.