Establishment of a Novel Permissive Cell Line for the Propagation of Hepatitis C Virus by Expression of MicroRNA miR122

Abstract

The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of "cured" cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics.

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication

“…Alanine Mutation at Ser-235 Reduced NS5A Hyperphosphorylation with a Concomitant Increase in NS5A Hypophosphorylation-To examine the effects of alanine mutations on NS5A phosphorylation without the interference from the HCV life cycle, single and combinatory alanine mutant constructs were made in the CMV-driven NS3-NS5A expression vectors and used to transfect the HEK293T cells that do not support HCV replication (26). Without the interference of the HCV life cycle in the HEK293T cells, the NS5A protein was expressed from all NS3-NS5A constructs at a similar level (Fig.…”

Virus Meets Host MicroRNA: the Destroyer, the Booster, the Hijacker

“…The molecular mechanism by which miR-122 facilitates replication is still unknown, but it is critical for the HCV life cycle. miR-122 expression is sufficient to convert nonhepatic cells to become HCV permissive (61,62). On the other hand, silencing of miR-122 with an antisense locked nucleic acid oligonucleotide leads to long-lasting suppression of HCV in infected primate models (63).…”

Isolation and Characterization of an Huh.7.5.1-Derived Cell Clone Highly Permissive to Hepatitis C Virus

“…These cells show viral titers of 10 4 -10 5 infectious units/ml of culture supernatant in the HCV-JFH1 infection system, and the virus can be serially passaged without loss of infectivity (8). Very recent studies have also shown other hepatic and nonhepatic cell lines that endogenously or that exogenously express higher miR-122 levels and support HCV infection (12)(13)(14)(15), although infection levels in these models are similar to or lower than those in Huh7-derived cells.…”