Sun X L, Johnson R B, Hockman M A, Wang Q M
Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.
Biochem Biophys Res Commun. 2000 Feb 24;268(3):798-803. doi: 10.1006/bbrc.2000.2120.
The 65 kDa RNA-dependent RNA polymerase (NS5B), encoded by the hepatitis C virus (HCV) genome, is a key component involved in viral replication. Here we provide the direct evidence that purified HCV polymerase catalyzed de novo RNA synthesis in a primer-independent manner using homopolymers and HCV RNA as templates. The enzyme could utilize both polyC and polyU as templates for de novo RNA synthesis, suggesting that NS5B specifically recognized pyrimidine bases for initiation. More importantly, NS5B also catalyzed de novo RNA synthesis with an HCV RNA template; the resulting nascent RNA products, smaller than the template used, contained ATP as the first nucleotide. These results indicate that the newly synthesized RNAs did not result from template self-priming and suggest that a replication initiation site in the HCV RNA genome is a uridylate.
丙型肝炎病毒（HCV）基因组编码的65 kDa RNA依赖性RNA聚合酶（NS5B）是参与病毒复制的关键成分。在此，我们提供了直接证据，即纯化的HCV聚合酶以同聚物和HCV RNA为模板，以不依赖引物的方式催化从头RNA合成。该酶可以利用聚C和聚U作为从头RNA合成的模板，这表明NS5B特异性识别嘧啶碱基进行起始。更重要的是，NS5B还以HCV RNA为模板催化从头RNA合成；产生的新生RNA产物比所用模板小，含有ATP作为第一个核苷酸。这些结果表明，新合成的RNA不是由模板自我引发产生的，提示HCV RNA基因组中的复制起始位点是尿苷酸。
