Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function
Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function
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DOI：
10.1073/pnas.0908365106
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发表时间：
2009-11-24
影响因子：
11.1
通讯作者：
Levrero, Massimo
中科院分区：
文献类型：
--
作者：
Belloni, Laura;Pollicino, Teresa;Levrero, Massimo
HBV cccDNA 是所有病毒 mRNA 转录的模板，作为稳定的附加体积聚在受感染细胞的细胞核中，通过组蛋白和非组蛋白病毒和细胞蛋白组织成微型染色体。使用基于 cccDNA 特异性染色质免疫沉淀 (ChIP) 的定量分析，我们之前已表明 HBV 微型染色体的转录受到 cccDNA 结合组蛋白的表观遗传变化的调节，并且 cccDNA 结合 H3/H4 组蛋白乙酰化状态的调节会影响 HBV 复制。我们现在证明细胞组蛋白乙酰转移酶 CBP、p300 和 PCAF/GCN5 以及组蛋白脱乙酰酶 HDAC1 和 hSirt1 均在体内招募到 cccDNA 上。我们还发现，HBV 复制细胞中产生的 HBx 调节蛋白被募集到 cccDNA 微型染色体上，并且 cccDNA 上 HBx 募集的动力学与 HBV 复制平行。正如预期的那样，不表达 HBx 的 HBV 突变体的复制受到损害，而外源表达的 HBx 反式互补了复制缺陷。 p300 募集严重受损，在复制 HBx 突变体的细胞中，cccDNA 结合的组蛋白迅速低乙酰化，而组蛋白脱乙酰酶 hSirt1 和 HDAC1 的募集增加并发生在较早的时间。最后，HBx 突变体 cccDNA 转录的 pgRNA 显着减少。总而言之，我们的结果进一步支持了影响 cccDNA 功能和 HBV 复制的复杂表观遗传事件网络的存在，并确定了 HBx 控制 HBV 复制的表观遗传机制（即防止 cccDNA 脱乙酰化）。
HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.