Development of a novel system to study hepatitis delta virus genome replication
Development of a novel system to study hepatitis delta virus genome replication
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DOI：
10.1128/jvi.79.13.8182-8188.2005
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发表时间：
2005-07-01
影响因子：
5.4
通讯作者：
Taylor, JM
中科院分区：
文献类型：
--
作者：
Chang, JH;Gudima, SO;Taylor, JM
丁型肝炎病毒（HDV）基因组复制需要病毒编码的小丁型蛋白（Ag）。在复制过程中，核苷酸序列的变化在HDV RNA上积累，导致无功能甚至抑制性的三角银的翻译。设计了一个复制系统，其中所有的δ Ag都有条件地从一个单独的和不变的来源提供。用编码小δ Ag的cDNA单拷贝稳定转染了一株人胚胎肾细胞，并在四环素（TET）控制下表达。接下来，通过转染无法表达δ Ag的突变RNA，在这些细胞中启动HDV基因组复制。因此，这种RNA的复制是由tet诱导的δ Ag控制的。在没有TET的情况下，有足够的δ Ag允许低水平的HDV复制，可以维持至少1年。当TET加入后，δ Ag和基因组RNA在2天内显著增加。这种细胞的克隆被命名为293-HDV， HDV RNA复制的爆发干扰了细胞周期。在2天内，G(1)/G(0)细胞相对于S和G(2)/M细胞增强了5倍，到6天，出现了广泛的细胞脱离和死亡。这些发现和正在进行的其他研究表明，这一实验系统具有潜在的应用价值。
Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (delta Ag). During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of delta Ag species that are nonfunctional or even inhibitory. A replication system was devised where all delta Ag was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small delta Ag, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express delta Ag. Thus, replication of this RNA was under control of the TET-inducible delta Ag. In the absence of TET, there was sufficient delta Ag to allow a low level of HDV replication that could be maintained for at least I year. When TET was added, both delta Ag and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G(1)/G(0) cells relative to both S and G(2)/M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.