Gray Kathleen S, Allen Robert D, Farrell Michael L, Forrest J Craig, Speck Samuel H
Emory Vaccine Center, 1462 Clifton Road, Suite 429, Atlanta, GA 30322, USA.
J Virol. 2009 Jan;83(1):314-28. doi: 10.1128/JVI.01444-08. Epub 2008 Oct 29.
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.
在对小鼠γ疱疹病毒68（MHV68）基因50编码的必需立即早期转录激活因子（RTA）的表达需求进行特征分析的过程中，构建了一种重组病毒，其中已知的基因50启动子被删除（G50pKO）。令人惊讶的是，G50pKO突变体保留了在允许的小鼠成纤维细胞中复制的能力，尽管其动力学比野生型MHV68慢。对从G50pKO感染的成纤维细胞制备的RNA进行5'-cDNA末端快速扩增分析，发现了一个新的上游转录起始位点，在允许细胞的野生型MHV68感染过程中也会利用该位点。此外，远端基因50/RTA转录起始位点上游的区域在允许的NIH 3T12成纤维细胞以及小鼠巨噬细胞系RAW 264.7中均表现出启动子活性。另外，在RAW 264.7细胞中，用脂多糖处理细胞可使远端基因50/RTA启动子的活性强烈上调（>20倍）。对卡波西肉瘤相关疱疹病毒和爱泼斯坦-巴尔病毒感染的B细胞系在病毒再激活诱导后制备的RNA进行逆转录酶PCR分析，也揭示了在已知转录起始位点上游起始的基因50/RTA转录本的存在。后者表明基因50/RTA转录起始的替代方式是小鼠和人类γ疱疹病毒之间保守的一种策略。用MHV68 G50pKO感染小鼠证明了这种突变病毒在脾脏和腹腔渗出细胞（PEC）中建立潜伏感染的能力。然而，G50pKO突变体无法从潜伏感染的脾细胞中重新激活病毒，并且从潜伏感染的PEC中重新激活也存在显著缺陷，这支持了一种模型，即近端基因50/RTA启动子在病毒从潜伏感染中重新激活，特别是从B细胞中重新激活的过程中起关键作用。最后，对近端和远端基因50/RTA转录起始位点上游区域的病毒基因组甲基化分析表明，远端启动子在体内部分甲基化，在MHV68潜伏感染的B细胞系中高度甲基化，这表明DNA甲基化可能在病毒潜伏期间使该启动子的活性沉默。
