Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee
Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee
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DOI：
10.1073/pnas.94.16.8738
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发表时间：
1997-08-05
影响因子：
11.1
通讯作者：
Bukh, J
中科院分区：
文献类型：
--
作者：
Yanagi, M;Purcell, RH;Bukh, J
我们成功地构建了H77菌株的稳定全长cDNA克隆我们设计了具有固定的5'和3'末端的盒式载体，并通过将通过长反转录酶-PCR扩增的整个ORF直接克隆到该载体中，一步构建了多个H77全长cDNA克隆，用RNA转录本直接肝内注射黑猩猩，检测了两个完整全长cDNA克隆的感染性，但未发现HCV复制的证据。对这些克隆和另外16个全长克隆的序列分析显示，7个克隆的多聚蛋白合成有缺陷，其余9个克隆与H77的共有序列相比在预测的多聚蛋白中有6-28个氨基酸突变。接下来，我们仅用两个连接步骤从四个全长cDNA克隆构建了共有嵌合体，将来自该共有克隆的RNA转录物注射到黑猩猩的肝脏中导致病毒复制，从黑猩猩中回收的病毒的序列与注射的RNA转录物的序列相同，这种稳定的感染性分子克隆应该是一个重要的工具，为发展更好地了解HCV的分子生物学和发病机制。
We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype la) of hepatitis C virus (HCV), We devised a cassette vector with fixed 5' and 3' termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase-PCR, directly into this vector, The infectivity of two complete full-length cDNA clones was tested bg the direct intrahepatic injection of a chimpanzee with RNA transcripts, However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6-28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps, Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication, The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts, This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.