Effect of Cell Growth on Hepatitis C Virus (HCV) Replication and a Mechanism of Cell Confluence-Based Inhibition of HCV RNA and Protein Expression

Abstract

An intimate relationship between hepatitis C virus (HCV) replication and the physiological state of the host liver cells has been reported. In particular, a highly reproducible and reversible inhibitory effect of high cell density on HCV replication was observed: high levels of HCV RNA and protein can be detected in actively growing cells but decline sharply when the replicon cells reach confluence. Arrested cell growth of confluent cells has been proposed to be responsible for the inhibitory effect. Indeed, other means of arresting cell growth have also been shown to inhibit HCV replication. Here, we report a detailed study of the effect of cell growth and confluence on HCV replication using a flow cytometry-based assay that is not biased against cytostasis and reduced cell number. Although we readily reproduced the inhibitory effect of cell confluence on HCV replication, we found no evidence of inhibition by serum starvation, which arrested cell growth as expected. In addition, we observed no inhibitory effect by agents that perturb the cell cycle. Instead, our results suggest that the reduced intracellular pools of nucleosides account for the suppression of HCV expression in confluent cells, possibly through the shutoff of the de novo nucleoside biosynthetic pathway when cells become confluent. Adding exogenous uridine and cytidine to the culture medium restored HCV replication and expression in confluent cells. These results suggest that cell growth arrest is not sufficient for HCV replicon inhibition and reveal a mechanism for HCV RNA inhibition by cell confluence.Hepatitis C virus (HCV) is a positive-strand RNA virus that infects more than 170 million people worldwide. Its infection mainly affects the liver and leads to both acute and chronic liver diseases including cirrhosis and hepatocellular carcinoma. HCV replication has proved to be a good model system for studies of virus-host cell interactions, as the virus often establishes a chronic infection that typifies the intricate relationship between a pathogen and its host.Research on HCV infection in vitro has been hampered by lack of an efficient cell culture-based production and infection system. The very recent reports of production of infectious HCV particles in tissue culture will hopefully break through this barrier and usher in a new era of HCV research (15,26,32). Studies of HCV RNA replication, however, received a great boost with the development of the HCV subgenomic replicon system a number of years ago (1, 16). Many important questions about the RNA replication of the virus were answered through use of the replicon cells. Adaptive mutations that permit efficient RNA replication in cultured hepatoma cells as well as nonhepatic cell lines have been defined (1, 13, 33); critical components of replication have been mapped and the data greatly complemented the limited in vivo data available previously (5, 12, 31). A novel mechanism by which HCV suppresses the innate antiviral responses to establish persistent replication has been uncovered (4). Th...

Modeling Subgenomic Hepatitis C Virus RNA Kinetics during Treatment with Alpha Interferon

“…It has been reported that the growth state of the host cell can have detectable effects on the replication rate of HCV replicons (14,23,27). Because we wanted to ensure that our HCV IFN-␣ inhibition experiments were performed under conditions that limited any nonspecific effects IFN-␣ might have on the host cell, we initially monitored the effect that increasing IFN-␣ doses (i.e., 100, 250, 500, and 1,000 U/ml) had on Huh7 clone B cell viability and growth ( Fig.…”

Hepatitis C Virus-Induced Cancer Stem Cell-Like Signatures in Cell Culture and Murine Tumor Xenografts

“…We examined the impact of HCV replicon expression on the status of reprogrammingand hepatic progenitor cell-related proteins using GS5 and FCA4 cell lines (7,14,37). The GS5 cells are derived from the Huh7.5 hepatoma cell line that permits efficient replication of HCV.…”

“…The cells harbor a geneticin (G418)-selectable subgenomic HCV-1b replicon that encodes nonstructural proteins NS3 through NS5B and a functionally active NS5A-green fluorescent protein (GFP) chimera (34,37). The intensity of the GFP in GS5 cells indicates the replication efficiency of the replicon RNA (37). The cells were cultured in the absence of G418, followed by treatment with IFN-␣ and cyclosporine.…”

“…FCA4 cells are derived from the Huh7 hepatoma cell line, and the sustained replication of a subgenomic HCV replicon in this cell line has been characterized previously (1,14). The GS5 cells are derived from the Huh7.5 cell line (interferon-cured Huh7 cells negative for the HCV replicon [7]) and express an HCV subgenomic replicon that encodes NS5A-green fluorescent protein (GFP) (37). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1ϫ penicillin-streptomycin, 1ϫ nonessential amino acids, and 10% fetal bovine serum (all from Invitrogen) and maintained at 37°C and 5% CO 2 .…”

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

“…Thus, higher expression levels of these genes may facilitate the growth of Huh7.5.1-8 cells. Because HCV replication is known to vary between stages of cell growth (42)(43)(44), further investigations of the 6 genes identified in the present study are required to determine their roles in the HCV life cycle, including RNA replication.…”