Hepatol. 58:583C592. general drug resistance phenotype to the computer virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the computer virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness. INTRODUCTION Selection of viral mutants resistant to antiviral brokers is a major problem for the successful treatment of viral diseases. In the case of RNA viruses, high mutation rates during genome replication provide viral populations with an ample reservoir of phenotypic variants, including mutants that can escape selective constraints. Resistance to a single drug that targets a viral protein develops at a rate that depends on the genetic barrier (number and types of mutations needed to acquire resistance) and the phenotypic barrier (fitness cost) imposed by the resistance mutations (1,C16). When drug resistance mutations do not entail a significant fitness costeither because the mutations do not critically affect viral functions or because compensatory mutations are acquiredthey may reach detectable levels despite no prior exposure of the viral populace to the drug (1, 16,C27). Control of hepatitis C computer virus (HCV) infections is usually hampered by the complexity of HCV quasispecies replicating in the liver (16, 28, 29). Directly acting antiviral brokers (DAAs)some currently in use as well as others under developmentoffer great promise for control of HCV either as a substitute for or complement of the standard-of-care (SOC) therapy based on treatment using a combination of pegylated alpha interferon (IFN-) and ribavirin (30,C36). Combinations that PF 4708671 PF 4708671 include the polymerase inhibitor sofosbuvir have produced sustained viral IEGF responses that in some cases have been higher than 90% in clinical trials (37,C40), but the possible impact of resistance mutations is not known; sofosbuvir resistance substitution S282T in NS5B is present in the sequence of HCV reference isolate ED43 of genotype 4a and L159F is present in the mutant spectrum of HCV quasispecies following treatment of HCV p100 with ribavirin (I. Gallego, E. Domingo, and C. Perales, unpublished results). The introduction of cell culture systems designed to achieve replication of full-length, infectious HCV (41,C43) has opened the way to studies on antiviral brokers for HCV in cell culture. Using this system (44), we performed up to 100 serial passages in the human hepatoma Huh-7.5 cell line, either in the absence or the presence of different concentrations of IFN- (45). In the course of these studies, we made the unexpected observations that populations of HCV passaged in the absence of IFN- acquired partial resistance to IFN- and that their capacity to shut off host cell protein synthesis was increased relative to that of the parental virus HCV p0 (where HCV p0 represents the HCV population before the first passage in Huh-7.5 cells) (45). It was unlikely that selection for partial IFN- resistance was due to endogenous IFN produced by the host cell since the Huh-7.5 cells used for the infections are defective in IFN production (46, PF 4708671 47). This observation raised three issues: (i) what the difference is in replicative parameters between HCV p0 and the passaged populations, (ii) whether the partial resistance is unique to IFN- or whether it extends to other anti-HCV drugs, and (iii) what the molecular basis is of either a specific or general partial resistance to drugs. We addressed these issues in the present study and show that passaged HCV displays increased replicative capacity and diminished sensitivity not only to IFN- but also to several other anti-HCV drugs. Furthermore, independent HCV evolutionary lineages and biological clones display the same behavior. Mutant spectrum analysis and viral replication in the absence and presence of drugs render unlikely the possibility that the presence of drug resistance mutations in the passaged populations is responsible for the expanded drug resistance. The results provide evidence that increased replicative HCV fitness results in a multidrug resistance phenotype. Implications for treatment of acute versus chronic HCV infections are discussed. MATERIALS AND METHODS Cells, viruses, and drugs. The origin of Huh-7.5, Huh-7 Lunet, and Huh-7.5 reporter cell lines and procedures for cell growth in Dulbecco’s modification of Eagle’s medium (DMEM) have been previously described (45, 48, 49); cells were cultured at 37C and 5%.