The understanding of molecular mechanism of hepatitis C virus (HCV) assembly has been frustrated because of lacking of productive cell culture system. Recently, a robust system for production of recombinant infectious...
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The understanding of molecular mechanism of hepatitis C virus (HCV) assembly has been frustrated because of lacking of productive cell culture system. Recently, a robust system for production of recombinant infectious HCV particle by using the JFH-1 isolate has been established. In this study, we applied this system to investigation of mechanism of virion assembly and examined whether trans-encapsidation of a subgenomic HCV replicon RNA lacking the structural-coding region is possible. Human hepatoma Huh-7 derived cell lines that constitutively express HCV JFH-1 structural proteins as well as the viral subgenomic RNA Were constructed. To partially purify putative HCV-like particles (HCV-LPs) from the culture supernatants of the cell lines, cultured media were subjected to sucrose cushion/density gradient ultracentrifugation and heparin affinity chromatography. We found that (1) the HCV-LPs can be purified by the heparin affinity column and (2) several spherical structures like matured HCV particles are detected by electronic microscopy, indicating that the established cell lines produce HCV-LPs and release them into the culture medium. To determine trans-encapsidation of the viral RNA into the HCV-LPs, CO-existence of the viral RNA and core protein in fractions of culture medium separated by density gradients were analyzed. Although distribution patterns of the viral RNA and core were not consistent, some CO-existence was found in several fractions such as 1.14 g/ml fraction. However, no or little infectivity of the HCV-LPs was observed in an infection experiment with permissive Huh-7-derived cells. To improve the trans-encapsidation system, development of new cell lines harboring replicon RNA including NS2 region is under way. The culture system with efficient production of HCV-LPs would be a valuable tool for studying the viral assembly and can be applied for the vaccine development.
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