Alternatively Initiated Gene 50/RTA Transcripts Expressed during Murine and Human Gammaherpesvirus Reactivation from Latency
Alternatively Initiated Gene 50/RTA Transcripts Expressed during Murine and Human Gammaherpesvirus Reactivation from Latency
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DOI：
10.1128/jvi.01444-08
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发表时间：
2009-01-01
影响因子：
5.4
通讯作者：
Speck, Samuel H.
中科院分区：
文献类型：
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作者：
Gray, Kathleen S.;Allen, Robert D., III;Speck, Samuel H.
在表征由鼠γ疱疹病毒68（MHV 68）基因50编码的必需的立即早期转录激活因子（RTA）的表达要求的过程中，产生了其中已知基因50启动子缺失的重组病毒（G50 pKO）。令人惊讶的是，G50 pKO突变体保留了在允许的鼠成纤维细胞中复制的能力，尽管动力学比野生型MHV 68慢。从G50 pKO感染的成纤维细胞制备的RNA的5 '-cDNA末端的快速扩增分析揭示了一个新的上游转录起始位点，该位点也在野生型MHV 68感染的允许细胞期间被利用。此外，远端基因50/RTA转录起始位点的上游区域在允许的NIH 3 T12成纤维细胞以及鼠巨噬细胞系RAW 264.7中都表现出启动子活性。此外，在RAW 264.7细胞中，通过用脂多糖处理细胞，远端基因50/RTA启动子的活性强烈上调（>20倍）。从卡波西肉瘤相关疱疹病毒和EB病毒感染的B细胞系制备的RNA的逆转录酶PCR分析，在诱导病毒再活化后，也揭示了基因50/RTA转录物的存在，其起始于已知转录起始位点的上游。后者认为，基因50/RTA转录的替代起始是鼠和人γ疱疹病毒中保守的策略。用MHV 68 G50 pKO感染小鼠证明了这种突变病毒在脾和腹膜渗出细胞（佩奇）中建立潜伏期的能力。然而，G50 pKO突变体不能从潜伏感染的脾细胞中再活化，并且还表现出从潜伏感染的佩奇中的显著再活化缺陷，这支持其中近端基因50/RTA启动子在病毒从潜伏（特别是从B细胞）中再活化中起关键作用的模型。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显示，远端启动子在体内部分甲基化，而在MHV 68潜伏感染的B细胞系中严重甲基化，这表明DNA甲基化可能有助于沉默病毒潜伏期间该启动子的活性。
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.