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.

Proteolytic processing of the Sindbis virus non-structural polyproteins (P123 and P1234) and synthesis of minusand plus-strand RNAs are highly regulated during virus infection. Although their precise roles have not been defined, these polyproteins, processing intermediates or mature cleavage products (nsPl -4) are believed to be essential components of viral replication and transcription complexes. In this study, we have shown that nsP4 can function as the polymerase for both minus-and plusstrand RNA synthesis. Mutations inactivating the nsP2 proteinase, resulting in uncleaved P123, led to enhanced accumulation of minus-strand RNAs and reduced accumulation of genomic and subgenomic plus-strand RNAs. In contrast, no RNA synthesis was observed with a mutation which increased the efficiency of P123 processing. Inclusion of this mutation in a P123 polyprotein with cleavage sites 1/2 and 2/3 blocked allowed synthesis of both minus-and plus-strand RNAs. We conclude that nsP4 and uncleaved P123 normally function as the minus-strand replication complex, and propose that processing of P123 switches the template preference of the complex to minus-strands, resulting in efficient synthesis of plus-strand genomic and subgenomic RNAs and shut-off of minus-strand RNA synthesis.

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn 2؉ than in the presence of Mg 2؉ . When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3 end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>50 M) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.Hepatitis C virus (HCV) is the major causative agent of non-A, non-B viral hepatitis (23). Although acute HCV infection is often asymptomatic, nearly 80% of cases resolve to chronic hepatitis, which may lead to progressive liver disease, such as cirrhosis, and liver failure. Chronic HCV infection is also associated with the development of hepatocellular carcinoma (23). It is estimated that 170 million people worldwide and more than 4 million people in the United States are currently infected with HCV (2, 65). Obviously, HCV infection remains a major threat to the public health all over the world.HCV is an enveloped RNA virus containing a singlestranded positive-sense RNA genome approximately 9.5 kb in length (14,31,56). The RNA genome consists of a 5Ј-untranslated region (5Ј UTR) of 341 nucleotides (12, 13), a large open reading frame (ORF) encoding a single polypeptide of 3,010 to 3,040 amino acids (14, 31, 56), and a 3Ј-untranslated region (3Ј UTR) of variable length (33,57,66). HCV is similar in amino acid sequence and genome organization to flaviviruses and pestiviruses (41), and therefore HCV was classified ...

Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ϳ5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.Hepatitis C virus (HCV) is the major causative agent for non-A, non-B hepatitis worldwide, which affects more than 3% of the world population. HCV establishes chronic infections in a large percentage of infected individuals, increasing the risk for developing liver cirrhosis and, in some cases, hepatocellular carcinoma. Although the current standard of care for HCV infection involves the use of PEGylated interferon and ribavirin, a large proportion of patients fail to respond to this therapy, and treatment is associated with frequent and sometimes serious side effects (9). Given the limited efficacy of the current therapy, the development of safer and more effective therapies is of tremendous importance.HCV is a positive-strand RNA virus belonging to the family Flaviviridae. The HCV genome consists of a ϳ9.6-kb RNA with a large open reading frame encoding a polyprotein of ϳ3,010 amino acids. The polyprotein is cleaved co-and posttranslationally by both cellular and viral proteases into at least 10 different products (10, 11). The viral proteins required for RNA replication include NS3, NS4A, NS4B, NS5A, and NS5B (4, 19). NS3 consists of an amino-terminal protease domain required for the cleavage of the remaining nonstructural proteins and a carboxyl-terminal helicase/NTPase domain (8,11,30). NS4A serves as a cofactor for NS3 protease and helicase activities (8). NS4B is a...

The hepatitis C virus NS5A protein is an established and clinically validated target for antiviral intervention by small molecules. Characterizations are presented of compounds identified as potent inhibitors of HCV replication to provide insight into structural elements that interact with the NS5A protein. UV-activated cross linking and affinity isolation was performed with one series to probe the physical interaction between the inhibitors and the NS5A protein expressed in HCV replicon cells. Resistance mapping with the second series was used to determine the functional impact of specific inhibitor subdomains on the interaction with NS5A. The data provide evidence for a direct high-affinity interaction between these inhibitors and the NS5A protein, with the interaction dependent on inhibitor stereochemistry. The functional data supports a model of inhibition that implicates inhibitor binding by covalently combining distinct pharmacophores across an NS5A dimer interface to achieve maximal inhibition of HCV replication.