Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo
Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo
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DOI：
10.1128/jvi.74.4.2046-2051.2000
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发表时间：
2000-02-01
影响因子：
5.4
通讯作者：
Rice, CM
中科院分区：
文献类型：
--
作者：
Kolykhalov, AA;Mihalik, K;Rice, CM
丙型肝炎病毒（HCV）感染是一个广泛的主要人类健康问题，在这种病毒的研究的重大障碍包括缺乏一个可靠的组织培养系统和小动物模型。最近，我们构建了全长HCV cDNA克隆，并通过肝内注射体外转录的RNA成功地在两只黑猩猩中引发HCV感染（A，A，Kolykhalov et al.，Science 277：570-574，1997）。为了验证抗HCV治疗剂开发的潜在靶点，我们构建了该原型感染性克隆的六个突变衍生物。四个克隆含有消除NS 2 -3蛋白酶、NS 3 -4A丝氨酸蛋白酶、NS 3 NTR/解旋酶和NS 5 B聚合酶活性的点突变。另外两个克隆含有HCV基因组RNA 3'末端高度保守的98个碱基序列的全部或部分缺失。将这六个克隆的RNA转录物肝内注射到黑猩猩体内。在注射后8个月内没有检测到HCV感染的迹象。用非突变的RNA转录物接种同一动物导致生产性HCV感染，如病毒血症、血清丙氨酸氨基转移酶升高和HCV特异性血清转化所证明的。这些数据表明，这四种HCV编码的酶活性和保守的3'末端RNA元件对于体内生产性复制是必需的。
Hepatitis C virus (HCV) infection is a widespread major human health concern, Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection in two chimpanzees by intrahepatic injection of in vitro-transcribed RNA (A, A, Kolykhalov et al., Science 277:570-574, 1997). In order to validate potential targets for development of anti-HCV therapeutics, we constructed six mutant derivatives of this prototype infectious clone. Four clones contained point mutations ablating the activity of the NS2-3 protease, the NS3-4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highly conserved 98-base sequence at the 3' terminus of the HCV genome RNA. The RNA transcript from each of the six clones was injected intrahepatically into a chimpanzee. No signs of HCV infection were detected in the 8 months following the injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion, These data suggest that these four HCV-encoded enzymatic activities and the conserved 3' terminal RNA element are essential for productive replication in vivo.