In past years, anti-hepatitis C therapy has modestly improved; however, a currently available combination therapy, consisting of interferon and the nucleoside analog, ribavirin, shows a sustained response in only less than half of the treated patients. The development of innovative treatment alternatives for patients infected with HCV is urgently required, and a better understanding of the life cycle of HCV should allow us to improve HCV therapies. However, due to the lack of an in vitro cell culture system for the isolation of virus directly from patient sera at present, various surrogate systems such as replicon cells (Lohmann et al., 1999) , pseudotype viruses (Lagging et al., 1998; Matsuura et al., 2001; Bartosch et al., 2003; Hsu et al., 2003; Tani et al., 2007) , or trans-complement particles (Ishii et al., 2008; Steinmann et al., 2008) have been developed to study each step of HCV infection. Although in vitro binding assays using soluble purified envelope proteins or HCV-LPs have identified several candidate receptors for HCV, the final determination of a true entry receptor or coreceptor capable of internalizing HCV may be made using an infection assay. Toward this end, pseudotype virus systems based on VSV and retrovirus or lentivirus have been established and applied to identify entry receptors for HCV. Although it is still unknown how HCV envelope proteins retained in the endoplasmic reticulum (ER) are incorporated into both VSV and retroviruses, which naturally bud from the plasma membrane, significant infectivity of these pseudotype viruses has been exhibited in several human hepatoma cell lines. These infections could be inhibited by treatment with antibodies or soluble proteins against putative receptors or HCV envelope proteins, or by a knockdown of receptor molecules by small interfering RNAs (siRNAs), suggesting that innate HCV infection had occurred. We also successfully generated infectious pseudotype and recombinant VSVs incorporating unmodified HCV envelope proteins in hepatic and non-hepatic human cell lines. These viruses exhibited high infectivity in a human hepatoma cell line, Huh7, which is highly susceptible to infection by cell-cultured HCV (HCVcc). The recombinant virus, but not the pseudotype virus, was able to propagate and form foci only in Huh7 cells. The infection of Huh7 cells with pseudotype and recombinant viruses was inhibited by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. These viruses, as well as pseudotype retroviruses (HCVpp) or HCVcc, were sensitive to the inhibitors of vacuolar acidification, such as ammonium chloride, concanamycin A, or bafilomycin A 1 , or formation of clathrin-coated pits, chlorpromazine, suggesting that these viruses enter via pH-dependent and clathrin-mediated endocytosis into target cells (Blanchard et al., 2006; Tani et al., 2007) . The infectivity of the recombinant virus was inhibited by an ER αglucosidase inhibitor, N -(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin www.frontiersin.org (Tani et al., 2007) . Focus formation of the recombinant virus was also impaired by Nn-DNJ treatment. It was obvious that the appearance of infectious or non-infectious viruses was dependent on the cell type as a result of the infectivity of the recombinant viruses generated from various cell lines. Although the precise mechanisms of HCV assembly or budding that cause the differences in infectivity of viruses generated from different cell lines is still unclear, host cellular factors might be involved in the assembly or budding steps in the generation of infectious particles.