The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC(50)) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log(10) reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.

BMS-790052 is the most potent hepatitis C virus (HCV) inhibitor reported to date, with 50% effective concentrations (EC 50 s) of <50 pM against genotype 1 replicons. This exceptional potency translated to rapid viral load declines in a phase I clinical study. By targeting NS5A, BMS-790052 is distinct from most HCV inhibitors in clinical evaluation. As an initial step toward correlating in vitro and in vivo resistances, multiple cell lines and selective pressures were used to identify BMS-790052-resistant variants in genotype 1 replicons. Similarities and differences were observed between genotypes 1a and 1b. For genotype 1b, L31F/V, P32L, and Y93H/N were identified as primary resistance mutations. L23F, R30Q, and P58S acted as secondary resistance substitutions, enhancing the resistance of primary mutations but themselves not conferring resistance. For genotype 1a, more sites of resistance were identified, and substitutions at these sites (M28T, Q30E/H/R, L31M/V, P32L, and Y93C/H/N) conferred higher levels of resistance. For both subtypes, combining two resistance mutations markedly decreased inhibitor susceptibility. Selection studies with a 1b/1a hybrid replicon highlighted the importance of the NS5A N-terminal region in determining genotype-specific inhibitor responses. As single mutations, Q30E and Y93N in genotype 1a conferred the highest levels of resistance. For genotype 1b, BMS-790052 retained subnanomolar potency against all variants with single amino acid substitutions, suggesting that multiple mutations will likely be required for significant in vivo resistance in this genetic background. Importantly, BMS-790052-resistant variants remained fully sensitive to alpha interferon and small-molecule inhibitors of HCV protease and polymerase.Hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting up to 180 million people worldwide (24, 32). HCV is an enveloped, positive-strand RNA virus and is the sole member of the Hepacivirus genus of the Flaviviridae family (17). HCV is classified into six major genotypes, each with multiple subtypes, based on sequence diversity (17, 33). The current standard of care for HCV infection includes therapy with a combination of pegylated alpha interferon (pegIFN-␣) and ribavirin. This treatment is often associated with limiting side effects, and effectiveness is highly genotype dependent (3,30,38). For genotypes 1a and 1b, accounting for ϳ60% of global infections, long-term efficacy or a sustained virological response is achieved in only ϳ50% of chronically infected individuals (3, 24). An increasing number of small-molecule inhibitors targeting specific viral proteins are entering into clinical evaluation (1,25,27,30,38). Collectively, these inhibitors are often referred to as direct-acting antiviral agents (DAA). The most advanced of these inhibitors target enzymatic activities of the HCV nonstructural proteins NS3 (serine protease) and NS5B (RNA-dependent RNA polymerase) (1, 27). We recently described a potent inhibitor of HCV RNA replication, BMS-79005...

The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/ NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor ␣. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/hRIP binding retained full NES function even though this sequence preserved only a single leucine residue. In contrast, nonfunctional activation domain mutants that were unable to bind Rab/hRIP had also lost NES function. These data demonstrate that NES activity is a defining characteristic of the activation domains found in the Rev/Rex class of retroviral regulatory proteins and strongly support the hypothesis that the Rab/hRIP cofactor plays a critical role in mediating the biological activity of these NESs. In addition, these data suggest a consensus sequence for NESs of the Rev/Rex class.The pathogenic complex retroviruses human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) belong to distinct retroviral families and display little sequence homology. The approaches used by these viruses to regulate proviral gene expression are nevertheless remarkably similar (reviewed in reference 5). Thus, each virus encodes a transcriptional regulatory protein that acts on the respective viral long terminal repeat promoter element to dramatically enhance viral gene expression. In addition, each virus also encodes an essential posttranscriptional regulatory protein, termed Rex in HTLV-1 and Rev in HIV-1, that induces the sequence-specific nuclear export, and hence translation, of the incompletely spliced viral mRNA species that encode the various viral structural proteins (8,11,16,17,20,23,27).Initial evidence favoring the hypothesis that Rex and Rev, despite lacking any significant sequence identity, might nevertheless mediate the nuclear export of target RNAs via the same mechanism came from the finding that Rex could partly rescue the replication of a Rev-deficient HIV-1 provirus (36). Subsequently, both Rev and Rex were shown to bind directly to structured RNA response elements present in their target RNAs (3,6,14,19,29,38,41) and to contain multimerization domains that are essential for the recruitment of additional Rev or Rex molecules (Fig. 1) (2, 26, 34). In addition, both Rev and Rex contain short, leu...