Analysis of persistent infection mechanism of RNA virus

HCV基因组结构分析和病毒复制遗传分析的主要障碍之一是缺乏允许HCV复制的可靠细胞培养系统。因此，我们利用完整的HCV cDNA和T7 RNA聚合酶重组腺病毒系统建立了HCV复制系统。方法：采用腺病毒/T7 RNA聚合酶杂交表达系统瞬时表达推定的HCV全长基因组（1-9603）。我们用HCV全cDNA克隆转染HCV高敏感细胞系（IMY细胞）并感染重组腺病毒。用免疫电镜对HCV颗粒进行了表征。结果：转染后的HCV基因组在细胞内复制，HCV子代RNA的出现和负链病毒RNA的检测证实了这一点。免疫电镜研究表明，在转染的细胞和培养基中检测到HCV样颗粒。我们展示了HCV在细胞内的复制过程，阐明了病毒颗粒的结构。结论：该表达系统使用了HCV全cDNA克隆，显示了3'X区域对HCV在细胞内复制的重要性。电镜研究揭示了病毒核粒在细胞内的动态变化。此外，还证明了核心粒子可以折叠六边形结构。

One of the major impediments to the structural analysis of the HCV genome and genetic analysis of viral replication has been lack of a reliable cell culture system permissive for HCV replication. Therefore, we established the HCV replication system by using entire HCV cDNA and T7 RNA polymerase recombinant adeno virus system.Methods : the putative full-length HCV genome (1-9603) was transiently expressed by the adeno virus/T7 RNA polymerase hybrid expression system. We transfected to a HCV high sensitive cell line (IMY cell) with HCV entire cDNA clone and infected recombinant adeno virus. The HCV particle was characterized by immuno-electron microscopy. Results : The transfected HCV genome replicated in cells, as evidenced by appearance of progeny HCV RNA and detection of negative-strand viral RNA.Immuno-electron microscopic studies have shown that the HCV like-particle was detected in the transfected cells and culture medium. We show the replication process of HCV in the cell and clarified the structure of viral particle. Conclusion : The expression system using HCV entire cDNA clone and showed the importance of 3'X region for HCV replication in the cell. The elctron microscopy study revealed the dynamics of viral core particle in the cells. Moreover the core particle was proved to fold the hexagonal structure.