Children's Hospital, Laboratory for Molecular Biology, Charité Universitätsmedizin Berlin, Berlin, Germany.
PLoS Pathog. 2010 Sep 9;6(9):e1001096. doi: 10.1371/journal.ppat.1001096.
The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.
人类巨细胞病毒 (HCMV) 裂解感染的开始与宿主细胞周期严格同步。感染的 G0/G1 细胞支持病毒早期即刻 (IE) 基因表达，并进入 G1/S 边界，最终停滞。相比之下，S/G2 细胞可以被感染，但有效阻断 IE 基因表达，并且只有在宿主细胞分裂并重新进入 G1 后，这种抑制才会解除。在潜伏感染中，IE 基因表达也受到抑制，为了重新激活，必须克服对 IE 基因表达的这种抑制。人们对哪些病毒和/或细胞活动维持细胞周期或潜伏相关病毒 IE 基因抑制知之甚少，以及这两种机制是否可能相关。在这里，我们表明，S 期和 G2 期 IE 基因表达的阻断可以通过遗传毒性应激和细胞 DNA 复制的化学抑制剂来克服，这表明细胞周期依赖性信号通路参与了控制 IE 基因抑制。IE 表达的检查点依赖性拯救严格依赖于 p53，并且在没有检查点激活的情况下，通过依赖 p53 的蛋白酶体抑制来模拟。p53 下游细胞周期蛋白依赖性激酶 (CDK) 抑制剂 p21 的需求表明 CDK 在控制 S/G2 中的 IE 基因抑制中起关键作用，并且用 CDK 抑制剂 roscovitine 处理 S/G2 细胞可独立于 p53 缓解 IE 抑制。重要的是，CDK 抑制也克服了 NTera2 (NT2) 细胞静止感染中 IE 表达的阻断。因此，及时阻断 CDK 活性不仅确保了细胞周期依赖性 HCMV IE 基因表达程序的时相特异性，而且还在防止建立潜伏样状态方面发挥了迄今为止尚未认识到的作用。
