Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.
Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.
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DOI：
10.1371/journal.ppat.0030139
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发表时间：
2007-09-07
期刊：
影响因子：
6.7
通讯作者：
Lemon SM
中科院分区：
文献类型：
--
作者：
Munakata T;Liang Y;Kim S;McGivern DR;Huibregtse J;Nomoto A;Lemon SM
丙型肝炎病毒（HCV）是一种常引起持续性感染的正链RNA病毒，与肝细胞癌的发展有独特的相关性。虽然HCV病毒促进癌症的机制尚不清楚，但先前的研究表明，HCV RNA依赖的RNA聚合酶非结构蛋白5B （NS5B）与视网膜母细胞瘤肿瘤抑制蛋白（pRb）形成复合物，靶向其降解，激活e2f应答启动子，并刺激细胞增殖。在这里，我们描述了HCV调控pRb的机制及其与HCV感染的相关性。我们发现，在感染基因型1a或2a型HCV的培养肝癌细胞后，pRb的丰度被强烈下调，其正常的核定位改变为包括一个主要的细胞质成分。我们进一步证明，这是由于ns5b依赖的pRb泛素化及其随后通过蛋白酶体降解。ns5b依赖的pRb泛素化需要e6相关蛋白（E6AP）的泛素连接酶活性，因为在含有HCV RNA复制子的细胞中，通过siRNA敲低E6AP或过表达显性阴性E6AP突变体来恢复pRb的丰度。E6AP也以依赖ns5b的方式与pRb形成复合物。这些发现提示了一种新的调控pRb的机制，其中HCV NS5B蛋白将pRb捕获在细胞质中，随后在导致pRb泛素化的过程中将E6AP招募到该复合体中。感染HCV的细胞中pRb/E2F调控通路的破坏可能促进肝细胞增殖和染色体不稳定，这是肝癌发展的重要因素。感染丙型肝炎病毒（HCV）的人患肝癌的风险增加。这是值得注意的，因为丙型肝炎病毒是一种RNA病毒，其复制仅限于细胞质，没有将其基因组整合到宿主细胞DNA中的潜力。虽然慢性炎症可能导致肝癌，但先前对HCV转基因小鼠的研究表明，病毒蛋白本质上是致癌的。在这项研究中，我们研究了其中一种RNA依赖性RNA聚合酶非结构蛋白5B与一种重要的细胞肿瘤抑制蛋白视网膜母细胞瘤蛋白（pRb）的相互作用。pRb是细胞周期的主要调节器，它所调节的许多基因中的一些基因表达的改变可能导致癌症。我们发现，在感染HCV的细胞中，pRb的丰度被强烈下调，非结构蛋白5B通过细胞正常的蛋白质降解机制靶向pRb进行破坏。e6相关蛋白似乎在这一过程中发挥了作用，有趣的是，它还介导了另一种肿瘤抑制因子p53被乳头瘤病毒降解。hcv感染细胞中pRb功能的丧失可能促进肝细胞增殖以及染色体不稳定，这是肝癌发展的重要因素。
Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer. Persons infected with hepatitis C virus (HCV) are at increased risk for liver cancer. This is remarkable because HCV is an RNA virus with replication confined to the cytoplasm and no potential for integration of its genome into host cell DNA. While it is likely that chronic inflammation contributes to liver cancer, prior studies with HCV transgenic mice indicate that the viral proteins are intrinsically carcinogenic. In this study, we have examined the interaction of one of these, the RNA-dependent RNA polymerase nonstructural protein 5B, with an important cellular tumor suppressor protein, the retinoblastoma protein (pRb). pRb is a master regulator of the cell cycle, and altered expression of some of the many genes it regulates may lead to cancer. We show that the abundance of pRb is strongly downregulated in cells infected with HCV, and that nonstructural protein 5B targets pRb for destruction via the cell's normal protein degradation machinery. The E6-associated protein appears to play a role in this process, which is interesting as it also mediates the degradation of another tumor suppressor, p53, by papillomaviruses. The loss of pRb function in HCV-infected cells likely promotes hepatocellular proliferation as well chromosomal instability, factors important for the development of liver cancer.
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