Dynamics of Subgenomic Hepatitis C Virus Replicon RNA Levels in Huh-7 Cells after Exposure to Nucleoside Antimetabolites

Abstract

Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i) minimal interference with the exponential cell growth, (ii) minimal reduction in cellular host RNA levels, and (iii) reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.Despite the availability of infectious cDNA clones of the hepatitis C virus (HCV), efficient in vitro replication has not been observed (3). After transfection of subgenomic HCV RNA replicons that also express the neomycin phosphotransferase gene selection marker, HCV replication has been reported previously in the human hepatoma cell line Huh-7 (2, 21). Such HCV replicon-harboring cell lines could be cultivated for more than a year without signs of cytopathogenicity (26). High levels of HCV RNAs can be maintained in cells passaged under continuous selection with G418. In addition, high-level replication was reflected in the observed adaptations of the HCV replicon to the host cell (20).A tight coupling of the amount of intracellular HCV RNA and cell growth was observed. High levels of viral RNA were found in exponentially growing cells, but this was followed by a sharp decline in RNA levels when cells reached a confluent state (26,29). This suggests that cellular factors required for RNA replication and/or translation vary in abundance and become limited in resting cells. Several proteins have been suggested, but none of them has as yet been positively identified to be directly responsible (9,15,16,19,25,30). However, there are no reports in which the availability of the cellular nucleoside triphosphate pools were linked to the loss of steadystate level replication of HCV replicon RNA in confluent cells. It is well-known that confluent cells mainly depend on salvage nucleoside biosynthetic pathways, resulting in lowered concentrations of endogenous nucleoside pools. As most antimetabolite agents of the de novo nucleoside biosynthesis are known to chemically deplete nucleoside pools and induce a concentration-dependent cytostasis, evaluating these compounds against HCV replicon can shed light on the relationship between nucleoside pools and replicon dynamics. However, before doing so, a definition of specificity of antiviral ...

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

“…siRNAs targeting CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a tripartite enzyme that catalyzes the first three steps of pyrimidine biosynthesis, inhibited both the Luc-1b replicon and JFH1-2a virus expression. This activity is consistent with the known inhibitor of this enzyme, leflunomide, which has been shown previously to inhibit both respiratory syncytial virus and HCV (12,54). siRNAs targeting the mevalonate (diphospho) decarboxylase (MVD) enzyme, which catalyzes the formation of mevalonate, were found to inhibit Luc-1b replication (19).…”

Plural assay systems derived from different cell lines and hepatitis C virus strains are required for the objective evaluation of anti-hepatitis C virus reagents

“…In this class, we especially noticed that methotrexate (an anticancer drug) showed very strong anti-HCV activity (EC 50 0.1 M; CC 50 >200 M; SI >2000) in the ORL8 assay (upper panel in Fig. 1A and Table 1), whereas methotrexate showed very weak anti-HCV activity (EC 50 >200 M; CC 50 >200 M) in the OR6 assay as well as in a previous report [13] (upper panel in Fig. 1A and Table 1).…”

“…Although methotrexate showed very weak anti-HCV activity in the HuH-7-derived assay (Con-1 strain) used in a previous study [13] as well as in our OR6 and AH1R assays (O and AH1 strains), the ORL8 assay revealed very strong anti-HCV activity (SI >2000). Such drastic differences in both assays suggest that some host factor or factors required for HCV RNA replication are different between these two cell lines, although the anti-HCV target of methotrexate is unclear.…”

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

“…If estimates of 1,000-5,000 copies of replicon per stable replicon cell are accurate (24), the starting pool of 10 5 replicon cells in this assay contained more than 10 8 replicons. We previously showed that mutations resistant to 150 nM HCV-796 were present at a relative frequency of 1.1%, and that same concentration produced more than 800 distinct resistant foci in this assay.…”