ENT1, a Ribavirin Transporter, Plays a Pivotal Role in Antiviral Efficacy of Ribavirin in a Hepatitis C Virus Replication Cell System

Abstract

We previously showed that equilibrative nucleoside transporter 1 (ENT1) is a primary ribavirin transporter in human hepatocytes. However, because the role of this transporter in the antiviral mechanism of the drug remains unclear, the present study aimed to elucidate the role of ENT1 in ribavirin antiviral action. OR6 cells, a hepatitis C virus (HCV) replication system, were used to evaluate both ribavirin uptake and efficacy. The ribavirin transporter in OR6 cells was identified by mRNA expression analyses and transport assays. Nitrobenzylmercaptopurine riboside (NBMPR) and micro-RNA targeted to ENT1 mRNA (miR-ENT1) were used to reduce the ribavirin uptake level in OR6 cells. Our results showed that ribavirin antiviral activity was associated with its accumulation in OR6 cells, which was also closely associated with the uptake of the drug. It was found that the primary ribavirin transporter in OR6 cells was ENT1 and that inhibition of ENT1-mediated ribavirin uptake by NBMPR significantly attenuated the antiviral activity of the drug as well as its accumulation in OR6 cells. The results also showed that even a small reduction in the ENT1-mediated ribavirin uptake, achieved in this case using miR-ENT1, caused a significant decrease in its antiviral activity, thus indicating that the ENT1-mediated ribavirin uptake level determined its antiviral activity level in OR6 cells. In conclusion, our results show that by facilitating its uptake and accumulation in OR6 cells, ENT1 plays a pivotal role in the antiviral effectiveness of ribavirin and therefore provides an important insight into the efficacy of the drug in anti-HCV therapy.C hronic hepatitis C is a major cause of liver cirrhosis and hepatocellular carcinoma, and a combination of interferon-␣ (IFN-␣) and ribavirin is a standard anti-hepatitis C virus (HCV) therapy. Since the addition of ribavirin to IFN-␣ significantly improves the rate of sustained virologic response (SVR) (40 to 60% in genotype 1 patients) (5), the drug plays a key role in current anti-HCV therapy.Ribavirin, a purine nucleoside analog, is phosphorylated intracellularly to form mono-, di-, and tri-phosphates, which then accumulate within cells at high concentrations (4, 13). While the primary anti-HCV mechanisms of the drug are still under debate, it is considered likely that the important actions take place within the cells themselves, and several mechanisms have been proposed to explain what occurs there. These include inhibition of inosine monophosphate dehydrogenase (reviewed in references 4 and 7 and references therein). Additionally, a recent study revealed that ribavirin potentiates IFN-␣ action by augmenting IFN-stimulated induction of gene expression (16).Taking into consideration the above-mentioned mechanisms, it is reasonable to assume that the uptake of ribavirin into hepatocytes is a prerequisite for its antiviral activity. Since ribavirin is a hydrophilic molecule, import of the drug into cells requires host nucleoside transporters, which are divided into two families: equilibra...

Persistent Hepatitis C Virus Infection Impairs Ribavirin Antiviral Activity through Clathrin-Mediated Trafficking of Equilibrative Nucleoside Transporter 1

“…These results based on cell culture experiments are consistent with clinical findings, where it has been observed that individuals with high viral titers are more resistant to combination therapy with IFN-␣ and RBV (34). The mechanism of impaired RBV antiviral activity in persistently HCV-infected cells is related to the reduced expression of ENT1, an RBV transporter, and the reduced uptake of RBV is in agreement with the results of other research reported previously (14)(15)(16)(17).…”

“…It has been reported that RBV resistance mechanisms in stable HCV replicon cell lines are due to mutations in viral RNA or to defective expression of nucleoside transporters in cells (13,14). Other studies have found that RBV resistance in HCV-infected cell cultures is due to reduced RBV uptake and low expression levels of nucleoside transporters (15)(16)(17)(18). The contribution of cellular factors in RBV resistance mechanisms has been described in HCV and other virus infection models (18,19).…”

Impact of Solute Carrier Family 29 Member 1 (SLC29A1) Single Nucleotide Polymorphisms on mRNA Expression in Peripheral Blood Mononuclear Cells

“…14) SLC29A1 mRNA knockdown reduced RBV uptake level in OR6 cells. 15) Thus, mRNA expression is responsible for the transport activity of SLC29A1. Since higher mRNA expression in PBMC may produce higher intracellular uptake and gastrointestinal absorption of RBV, Tsubota's findings that patients with wildtype rs6932345 possessed higher blood RBV and responded well to IFN/RBV therapy might be relevant.…”

Understanding the molecular mechanism of host-based statin resistance in hepatitis C virus replicon containing cells

“…One mutation in NS5A (D320E) conferred a low-level resistance to alisporivir (3.9-fold), probably by reducing the need for cyclophilin A-dependent isomerisation of NS5A. On the other hand, ribavirinresistant HCV replicon cells proved to have a cellular resistance mechanism Page 5 of 73 A c c e p t e d M a n u s c r i p t 5 consisting of a decreased uptake of ribavirin by ENT1 transporters [20]. Here, we wanted to determine whether fluvastatin-resistant HCV replicon containing cells can be selected.…”