The pre-S region determines the intracellular localization and appearance of hepatitis B virus
The pre-S region determines the intracellular localization and appearance of hepatitis B virus
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DOI：
10.1002/hep.510300206
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发表时间：
1999-08-01
期刊：
影响因子：
13.5
通讯作者：
Trautwein, C
中科院分区：
文献类型：
--
作者：
Bock, CT;Tillmann, HL;Trautwein, C
乙型肝炎病毒 (HBV) 前 S 区对于病毒组装和外观的功能作用尚不完全清楚。在这项研究中，研究了 3 个自然发生的突变体。前S区的三个突变体——CCAAT盒中的点突变（MUT1）、CCAAT盒3'端的6-bp缺失（MUT2）和preS2结构域中的153-bp缺失（MUT3）——被单独克隆或组合克隆到具有复制能力的HBV质粒中，并转染到肝癌细胞中。通过 Northern Blot、引物延伸分析、免疫荧光研究、酶联免疫吸附测定和电子显微镜研究了对 HBV 组装和外观的影响。当质粒中包含 MUT1 或 -2 时，发现前 S/S mRNA 转录物与野生型 (wt) HBV 的比例呈倒数。两种突变体的细胞内定位显示大S蛋白在内质网中保留并且核心蛋白在核中积累。 MUT1和-2或其中包含1个突变体的组合减少了S蛋白的胞外量。然而，病毒产物的细胞外外观与 wtHBV 相当。相比之下，MUT3 则出现了重大变化。病毒颗粒具有煎蛋般的外观，细丝具有螺旋状的外观。 S 启动子突变 MUT1 和 MUT2 与病毒滞留相关。 MUT3 会导致病毒颗粒畸形。因此，前S结构域的不同区域对于确定HBV的细胞内定位和细胞外外观至关重要，并且可能有助于慢性HBV感染的预后。
The functional role of the hepatitis B virus (HBV) pre-S region for assembly and appearance of the virus is not completely understood. In this study, 3 natural-occurring mutants were investigated. Three mutants of the pre-S region-a point mutation in the CCAAT box (MUT1), a 6-bp deletion (MUT2) 3' of the CCAAT box, and a 153-bp deletion (MUT3) in the preS2 domain-were cloned alone or in combinations in replication-competent HBV plasmids and transfected in hepatoma cells. The impact on HBV assembly and appearance was studied by Northern Blot, primer extension analysis, immunofluorescence studies, enzyme-linked immunosorbent assay, and electron microscopy. An inversed ratio of pre-S/S mRNA transcripts compared with wild-type (wt) HBV was found when either MUT1 or -2 were included into the plasmid. Intracellular localization with both mutants showed retention of large S-protein in the endoplasmic reticulum and nuclear accumulation of core protein. The extracellular amount of S-protein was reduced with MUT1 and -2 or combinations in which 1 of the mutants was included. However, the extracellular appearance of viral products was comparable with wtHBV. In contrast, MUT3 showed major changes. Virion-like particles had a fried-egg, and filaments a screw-like appearance. The S-promoter mutations MUT1 and MUT2 correlated with viral retention. MUT3 leads to malformed viral particles. Therefore, different regions in the pre-S domain are essential to determine the intracellular localization and extracellular appearance of HBV, and might contribute to the prognosis of chronic HBV infection.