MicroRNA-122-dependent and -independent replication of Hepatitis C Virus in Hep3B human hepatoma cells

Abstract

The study of Hepatitis C Virus (HCV) has benefitted from the use of the Huh7 cell culture system, but until recently there were no other widely used alternatives to this cell line. Here we render another human hepatoma cell line, Hep3B, permissive to the complete virus life cycle by supplementation with the liver-specific microRNA miR-122, known to aid HCV RNA accumulation. When supplemented, Hep3B cells produce J6/JFH-1 virus titres indistinguishable from those produced by Huh7.5 cells. Interestingly, we were able to detect and characterize miR-122-independent replication of di-cistronic replicons in Hep3B cells. Further, we show that Argonaute-2 (Ago2) is required for miR-122-dependent replication, but dispensable for miR-122-independent replication, confirming Ago2's role in mediating the activity of miR-122. Thus Hep3B cells are a model system for the study of HCV, and miR-122 independent replication is a model to identify proteins involved in the function of miR-122.

Regulation of Hepatitis C Virus Genome Replication by Xrn1 and MicroRNA-122 Binding to Individual Sites in the 5′ Untranslated Region

“…Plasmids pSGR JFH-1 Fluc WT and pSGR JFH-1 Fluc GND contain bicistronic JFH-1-derived subgenomic replicon (SGR) cDNAs with a firefly luciferase reporter; the GDD-to-GND mutation renders the viral polymerase nonfunctional (22). pSGR JFH-1 S1:p3 Fluc WT, pSGR JFH-1 S2:p3 Fluc WT, and pSGR JFH-1 S1ϩS2:p3 Fluc WT contain C-to-G mutations at position 3 of miR-122 binding site 1, site 2, or sites 1 and 2, respectively, generated as described by Thibault et al (4). Plasmids pJ6/JFH-1 FL Rluc WT and pJ6/JFH-1 FL Rluc GNN, also known as pJ6/JFH-1(p7Rluc2A) and pJ6/JFH-1(p7Rluc2A), were obtained from C. M. Rice.…”

“…Plasmids pT7 luciferase (containing firefly luciferase; Promega, Nepean, ON, Canada) and pRL-TK (containing Renilla luciferase; Promega) were used as the templates for production of mRNA. Viral RNA and mRNA were in vitro transcribed from these plasmids as described by Thibault et al (4).…”

“…One of the major determinants of the virus' hepatotropism is its requirement for the liver-specific, liver-abundant miR-122 microRNA (miRNA) (3,4). miR-122 binds to two sites at the 5= end of the virus' positive-sense RNA genome and has been shown to directly enhance viral RNA accumulation, since mutation of the miR-122 binding sites abolishes RNA accumulation, and the provision of exogenous miR-122 sequences that have compensatory mutations to restore binding also reinstates RNA accumulation (4)(5)(6)(7)(8)(9)(10).…”

“…miR-122 binds to two sites at the 5= end of the virus' positive-sense RNA genome and has been shown to directly enhance viral RNA accumulation, since mutation of the miR-122 binding sites abolishes RNA accumulation, and the provision of exogenous miR-122 sequences that have compensatory mutations to restore binding also reinstates RNA accumulation (4)(5)(6)(7)(8)(9)(10). Argonaute-2, one of the key effector proteins in the microRNA pathway and a component of the RNA-induced silencing complex (RISC), binds in association with miR-122 and is required to increase HCV replication, while several other proteins in the microRNA pathway and RISC have been implicated in either the biogenesis or activity of miR-122 (5,(11)(12)(13)(14).…”

“…Although miR-122 uses canonical microRNA seed sequence binding and RISC components when interacting with the HCV genome, it also binds to HCV nucleotides outside the seed sequence, creating a double-stranded RNA-protein structure that overhangs the 5= end of the viral genome, and also interacts with the "spacer" sequence located between miR-122 binding site 1 (S1) and S2 on the HCV 5= untranslated region (UTR) (7,11). We and others have ruled out any significant indirect influence of miR-122 on HCV in cell culture models; miR-122-mediated regulation of the cholesterol biosynthesis pathway had no significant effect on HCV RNA accumulation, and miR-122 binding mutant viral RNAs do not respond to wild-type (WT) miR-122 but will respond to mutant miRNAs the same as wild-type HCV responds to miR-122 (4,6,9).…”