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...

SummaryIn bacteriophage l, formation of a transcriptional anti-termination complex involving the elongating RNA polymerase is mediated by the interaction of boxB RNA with the RNA-binding domain of the N protein (N peptide). In an attempt to understand the spatial requirements for boxB/N peptide interaction within the anti-termination complex, the effects of changes in the distance between boxA and boxB RNA, the length of the boxB stem, and the distance between the N peptide and remainder of the N protein were examined using a bacterial reporter system. It was found that the requirements for boxB stem length and the distance between N peptide and the remainder of N were optimized and strict. In contrast, replacement of the boxB/N interaction by heterologous RNA-peptide interactions appeared to relax the strict requirement for RNA stem length and the orientation of the RNA-binding peptide, presumably due to the absence of the cooperative interaction between boxB/N and the host factor NusA. In addition, the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer. A further understanding of the structural organization of the anti-termination complex may provide insights into how functional ribonucleoprotein complexes may be engineered.

Human equilibrative nucleoside transporter 1 (hENT1) transports various nucleoside analogues into cells. Although the single hENT1 promoter region (P1) and the mRNA isoform (a1) have been characterized previously, we have recently identified additional promoter regions P2 and P3 (which primarily generate c1/2/3 mRNAs and d1/2/3/4 mRNAs, respectively) in the human liver. Therefore, this study aimed at identifying the primary hENT1 mRNA isoforms expressed in human hepatocytes, while simultaneously obtaining functional evidence of alternative hENT1 promoter usage. Our results showed that the expressions of hENT1c1, d3, and (to a lesser extent) c2 mRNAs were strikingly predominant over the other mRNA isoforms in human hepatocytes, that the abundant expression of these mRNAs was consistent with the high levels of P2 and P3 promoter activity, and that these promoters were significantly marked by transcriptionally active histone modification in hepatic cells. To summarize, our results demonstrate that, resulting from the manipulated alternative promoter usage, hENT1c1 and d3 (and c2) mRNAs are primarily expressed in human hepatocytes, which suggests that they may play important roles in controlling hENT1 expression levels in those cells. Our findings are expected to provide significant insights into the molecular machinery of hENT1 expression control.