Reduced Ribavirin Antiviral Efficacy via Nucleoside Transporter-Mediated Drug Resistance

Abstract

Treatment for hepatitis C virus infection currently consists of pegylated interferon and ribavirin (RBV), a nucleoside analog. Although RBV clearly plays a role in aiding the treatment response, its antiviral mechanism is unclear. Regardless of the specific mechanism of RBV, we hypothesize that differences in levels of cellular uptake of RBV may affect antiviral efficacy and treatment success and that cells may become RBV resistant through reduced uptake. We monitored RBV uptake in various cell lines and determined the effect of uptake capacity on viral replication. RBV-resistant cells demonstrated reduced RBV uptake and increased growth of a model RNA virus, poliovirus, in the presence of RBV. Overexpression of equilibrative nucleoside transporter 1 (ENT1) or concentrative nucleoside transporter 3 (CNT3) increased RBV uptake in RBV-sensitive cell lines and restored the uptake defect in most RBV-resistant cell lines. However, CNT3 is not expressed in Huh-7 liver cells, and inhibition of concentrative transport did not affect RBV uptake. Blocking equilibrative transport using the inhibitor nitrobenzylmercaptopurine riboside recapitulated the RBV-resistant phenotype in RBVsensitive cell lines, with a reduction in RBV uptake and increased poliovirus growth. Taken together, these results indicate that RBV uptake is restricted primarily to ENT1 in the cell lines examined. Interestingly, some RBV-resistant cell lines may compensate for reduced ENT1-mediated nucleoside uptake by increasing the activity of an alternative nucleoside transporter, ENT2. It is possible that RBV uptake affects the antiviral treatment response, either through natural differences in patients or through acquired resistance.Approximately 170 million people are infected with hepatitis C virus (HCV), with the majority developing chronic infection (1). With no vaccine currently available, the only approved treatment consists of a combination of alpha interferon (IFN-␣) and ribavirin (RBV), a guanosine nucleoside analog. IFN-␣ monotherapy has limited success, with only 16 to 20% of genotype 1-infected patients achieving a sustained virological response (SVR). However, the addition of RBV doubled response rates to 35 to 40%. Current treatment regimens including pegylated IFN and RBV achieve SVR rates of 54 to 56% in genotype 1-infected patients, while SVR rates of 70 to 80% are achieved in genotype 2-or 3-infected patients. The patient response is divided into three categories: SVR, end-of-treatment response and relapse, and nonresponse. Little is known about factors that influence the treatment response, although various host and viral factors have been implicated. For instance, genotype 1 infections are more difficult to treat than those of other genotypes. Additionally, male gender, AfricanAmerican race, advanced age, fibrosis, obesity, human immunodeficiency virus coinfection, and low RBV serum concentrations have been negatively correlated with treatment success (3,14,26,27,34,35).Although RBV clearly plays a role in the HCV treatment response, ...

Host-Based Ribavirin Resistance Influences Hepatitis C Virus Replication and Treatment Response

“…Nucleoside transporter transfections were conducted as previously described using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendation (27). Plasmid DNA (pcDNA 3.1, pENT1, and pCNT3) was provided by K. Giacomini (UCSF).…”

“…Total RNA was isolated from RBV R and RBV S Huh7.5 cells as previously described (27). The relative levels of ENT1 and glyceraldehyde-3-phosphate dehyrdrogenase (GAPDH) RNA were quantified using one-step real-time reverse transcriptase PCR (RT-PCR) and the comparative method as described previously (27).…”

“…Viral titers were determined using a focus-forming assay (10). Mahoney serotype 1 poliovirus was generated in HeLa cells as previously described (27).…”

“…The [ 3 H]RBV uptake assay was conducted as described previously (27). Cells were treated with medium containing 5 M RBV (containing approximately 1% [ 3 H]RBV as a tracer; Moraveck, Brea, CA) for 30 min.…”

“…Our previous work determined that ENT1 was the primary RBV transporter in Huh7 liver cells. RBV-resistant (RBV R ) Huh7 cells were generated by passage in increasing concentrations of RBV, and resistance was mediated by reduced RBV transport through ENT1 (27). As a result of decreased RBV import, RBV R Huh7 cells supported robust poliovirus replication in the presence of RBV compared to RBV-sensitive (RBV S ) Huh7 cells, suggesting that the level of RBV import impacts antiviral efficacy.…”