Identification of the key molecules for hepatitis C virus (HCV) replication and the translation research for the noble protocols against HCV using cell-based HCV replication model.

利用重组腺病毒载体和T7稳定细胞系，建立了基于细胞的丙型肝炎病毒（HCV）复制系统。在pH77和Ad-T7pol的作用下，HCV阳性链和阴性链RNA在感染后的前2至3天表达最强，此后表达减弱，但在9天内表达。这种HCV-RNA的持续表达也见于T7稳定细胞系的系统中。在这些系统中，干扰素（IFN）抑制HCV复制，并上调蛋白激酶R （PKR）途径。干扰素和利巴韦林（RBV）联合治疗是目前慢性丙型肝炎病毒抗病毒治疗的标准。在ifn诱导的蛋白中，PKR被认为是消除HCV的关键分子。另一方面，据报道，白细胞介素-8 （IL-8）可以抑制ifn诱导的抗病毒反应。在我们的复制模型中，HCV表达和IFN都能有效地诱导PKR及其调控蛋白。PKR直接调控HCV蛋白的表达。从我们的实验中，PKR直接抗病毒HCV，并且是IFN对HCV作用的中心介质。在我们的系统中，IL-8是由IFN和RBV共同诱导的。我们推测两种药物诱导IL-8可能不能产生足够的抗病毒作用。相反，增加的IL-8可能作为免疫调节剂或趋化因子在募集消除HCV的细胞中起间接作用。此外，我们通过收集IFN和RBV联合治疗的慢性丙型肝炎患者的外周血T细胞进行了体内实验。在早期病毒反应（EVR）患者中，HCV-RNA在12周内无法检测到，T细胞中的PKR mRNA明显高于非EVR患者。从这些结果中，我们得出结论，PKR和IL-8将是HCV复制和HCV持久性的关键分子。此外，我们必须考虑建立有效上调PKR的方案，以获得更强的抗hcv治疗。

We established the cell-based hepatitis C virus (HCV) replication system with recombinant adenovirus vectors and with T7 stable cell lines. With pH77 and Ad-T7pol, HCV positive and negative strand RNA expression was strongest in the first 2 to 3 days post-infection and diminished thereafter, but were expressed throughout the 9 days. This sustained expression of HCV-RNA was also seen in the system with T7 stable cell lines. In these systems, interferon (IFN) inhibits HCV replication, and upregulates the protein kinase R (PKR) pathway.IFN and ribavirin (RBV) combination therapy is the current standard of antiviral therapy for chronic HCV. Among IFN-induced proteins, PKR is considered as a key molecule to eliminate HCV. On the other hand, interleukin-8 (IL-8) has been reported to inhibit IFN-induced antiviral responses. In our replication model, both HCV expression and IFN, induced PKR and its regulated proteins effectively. HCV protein expression in turn was directly regulated by PKR. From our experiments, PKR is directly antiviral for HCV, and is a central mediator of IFN' s effects against HCV. About IL-8, it was induced by both IFN and RBV in our systems. We speculate that IL-8 induction by both agents might not produce sufficiently antiviral effects. Rather, increased IL-8 might have an indirect role as an immunomodulator or as a chemokine in recruitment of cells that eliminate HCV.Additionally, we did in vivo assay by collecting peripheral blood T cells from the chronic hepatitis C patients who were treated by IFN and RBV combination therapy. In the patients with early viral response (EVR), HCV-RNA become undetectable within 12 weeks, PKR mRNA in T cells are significantly higher than that in the patients with non-EVR.From these results, we concluded that PKR and IL-8 will be key molecules for HCV replication as well as HCV persistence. Moreover, we have to consider to establish the protocol which can upregulate PKR effectively for the stronger anti-HCV therapy.