Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein
Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein
复制标题
DOI：
10.1128/jvi.05423-11
复制
发表时间：
2011-12-01
影响因子：
5.4
通讯作者：
Branza-Nichita, Norica
中科院分区：
文献类型：
--
作者：
Dorobantu, Cristina;Macovei, Alina;Branza-Nichita, Norica
以前的报道表明，胆固醇耗尽的膜包膜的B型肝炎病毒（HBV）损害病毒感染的靶细胞。这种脂质在病毒生命周期后期的潜在功能仍然存在争议，病毒体和亚病毒颗粒（SVP）的分泌受到抑制或不受影响，这取决于用于降低细胞内胆固醇水平的实验方法。这项工作解决了宿主细胞胆固醇对HBV复制，组装和分泌的作用，使用另一种方法来抑制参与生物合成途径的酶。用脂蛋白去除血清（LPDS）培养HBV产生细胞导致治疗24小时内胆固醇量显著减少（约40%）。细胞暴露于氯丙嗪（一种由低密度脂蛋白受体用于内吞作用的网格蛋白介导途径的抑制剂）也影响胆固醇水平;然而，当使用合成抑制剂洛伐他汀时，这种抑制水平无法实现。在胆固醇耗尽的细胞中，HBV分泌被显著抑制（约80%），而SVP释放不受影响。病毒DNA基因组在LPDS处理的细胞中以时间依赖性方式积累。核衣壳和成熟病毒体的特异性免疫沉淀显示裸露核衣壳的量增加，而包膜蛋白的合成正常发生。在纯化的微粒体中的大包膜蛋白构象的分析之后，我们得出结论，胆固醇在维持这种多肽的双重拓扑结构中是重要的，这对于病毒的复制是至关重要的。
Previous reports have shown that cholesterol depletion of the membrane envelope of the hepatitis B virus (HBV) impairs viral infection of target cells. A potential function of this lipid in later steps of the viral life cycle remained controversial, with secretion of virions and subviral particles (SVP) being either inhibited or not affected, depending on the experimental approach employed to decrease the intracellular cholesterol level. This work addressed the role of host cell cholesterol on HBV replication, assembly, and secretion, using an alternative method to inhibition of the enzymes involved in the biosynthesis pathway. Growing HBV-producing cells with lipoprotein-depleted serum (LPDS) resulted in an important reduction of the amount of cholesterol within 24 h of treatment (about 40%). Cell exposure to chlorpromazine, an inhibitor of the clathrin-mediated pathway used by the low-density lipoprotein receptor for endocytosis, also impacted the cholesterol level; however, this level of inhibition was not achievable when the synthesis inhibitor lovastatin was used. HBV secretion was significantly inhibited in cholesterol-depleted cells (by similar to 80%), while SVP release remained unaffected. The viral DNA genome accumulated in LPDS-treated cells in a time-dependent manner. Specific immunoprecipitation of nucleocapsids and mature virions revealed an increased amount of naked nucleocapsids, while synthesis of the envelope proteins occurred as normally. Following analysis of the large envelope protein conformation in purified microsomes, we concluded that cholesterol is important in maintaining the dual topology of this polypeptide, which is critical for viral envelopment.