HepG2 Cells Expressing MicroRNA miR-122 Support the Entire Hepatitis C Virus Life Cycle

Abstract

The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.Hepatitis C virus (HCV), a member of the family Flaviviridae, is associated with more than half of newly diagnosed hepatocellular carcinomas in the United States (1, 31) and is the leading cause of liver transplants worldwide (7). Historically, a lack of model systems to study HCV replication has slowed the progress of HCV research and the development of specific antivirals. Few HCV genomes replicate in culture, and only one efficiently produces infectious particles (23,39,42). Most model systems rely on the Huh-7 cell line and derivatives, such as the highly HCV-permissive Huh-7.5 subclone (6). The development of novel cell systems capable of supporting the entire HCV life cycle would allow broader examination of interactions between HCV and host cell biology and would provide essential tools for anti-HCV drug discovery.MicroRNAs (miRNAs) are small RNA molecules predicted to downregulate the expression of one-third of human genes by reducing mRNA stability and/or translation, depending on the degree of complementarity between the miRNA and the mRNA target site (2). miR-122, a liver-specific miRNA expressed at high levels in hepatocytes, is required to support HCV RNA replication (19). Two miR-122 target sites have been identified in the 5Ј untranslated region of the HCV genome, and mutation of these sites disrupts HCV RNA replication. Replication is restored by cotransfection with RNA duplexes that produce a mutant miR-122 with complementary changes (16)(17)(18)(19)26). How miR-122 enhances HCV replication is unclear. Although its binding appears to enhance HCV internal ribosome entry site (IRES)-directed translation, this effect is not great enough to account for the difference in RNA replication (16,17,40). Furthermore, the normal cellular function of miR-122 in regulating cholesterol and fatty acid biosynthesis is not required for this enhancement (18). Thus, miR-122 has been hypothesized to directly affect HCV RNA replication or stability (17,19,26). Although inhibiting miR-122 has been shown to be an effective therapy in chimpanzees infected with HCV (21), further development of such therapies will require a firmer understanding of how miR-122 promotes HCV replication.The majority of published studies on the role of miR-122 in the HCV life cycle have described research conducted with HCV-permissive Huh-7 cells or derivatives, which express endogenous miR-122 (19). Two studies explored the effect of m...

MiR-122 in hepatic function and liver diseases

“…Chang et al (2008) showed that miR-122 could also enhance HCV replication in nonhepatic human embryonic kidney epithelial cells (HEK-293). Interestingly, expression of miR-122 has been shown to endow the ability of supporting efficient HCV RNA replication and infectious virion production in HepG2 cells (Narbus et al, 2011). Henke et al (2008) further uncovered a unique interaction of miR-122 with two binding sites in the 5′-UTR of the viral genome, which stimulates HCV translation by enhancing the association of ribosomes with the viral RNA.…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…First of all, their poor or absent polarization biases HCV entry studies. New models such as the partially polarized and HCV-permissive HepG2 CD81/miR-122 cell line might help tackling this concern [112]. Furthermore, despite some controversy, it is generally accepted that the VLDL secretion is altered in the Huh-7 cell line [28,166].…”

The role and clinical implications of microRNAs in hepatocellular carcinoma

“…miR-122 antagomir can inhibit replication of HCV, abundance of HCV core RNA, and expression of HCV nonstructural proteins (NS3 in CNS3 cells and NS3-5B in 9-13 replicon cells), in a time-and dosedependent manner [51,[60][61][62][63]. These findings are suggestive of miR-122 as a potential target for antiviral interventions in HCV patients [37,57,[64][65][66][67].…”

“…It is required for efficient replication of HCV in vitro and in vivo. Expression of miR-122 in HepG2 cells leads to efficient HCV RNA replication and infectious virion production [37]. When a missing HCV receptor was coexpressed with miR-122, HepG2 cells could efficiently facilitate viral entry and the entire life cycle of HCV.…”

“…Overexpression of miR-29a enhances migration of HepG2 cells [37]. Moreover, specific miR-29a inhibitors partially abolish HepG2-X cell migration without affecting HBx expression [38].…”