[Show abstract][Hide abstract] ABSTRACT: While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict emergence of drug resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con 1) replicon with an EC50 of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.
[Show abstract][Hide abstract] ABSTRACT: Cleavage of the hepatitis C virus (HCV) polyprotein by the viral NS3 protease releases functional viral proteins essential
for viral replication. Recent studies by Foy and coworkers strongly suggest that NS3-mediated cleavage of host factors may
abrogate cellular response to alpha interferon (IFN-α) (E. Foy, K. Li, R. Sumpter, Jr., Y.-M. Loo, C. L. Johnson, C. Wang,
P. M. Fish, M. Yoneyama, T. Fujita, S. M. Lemon, and M. Gale, Jr., Proc. Natl. Acad. Sci. USA 102:2986-2991, 2005, and E. Foy, K. Li, C. Wang, R. Sumpter, Jr., M. Ikeda, S. M. Lemon, and M. Gale, Jr., Science 300:1145-1148, 2003). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression
of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide
inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition
of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored
by immunofluorescence and real-time PCR analysis. Continuous exposure of replicon-bearing cell lines to six times the 90%
effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination
of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion
of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.
[Show abstract][Hide abstract] ABSTRACT: Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PP(i)) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PP(i), using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D(pol)) and the hepatitis C virus nonstructural protein 5B (NS5B) RdRp reactions. Light production was dependent on RdRp and sensitive to the concentration of oligonucleotide primer directing RNA synthesis. The assay system was found to be amenable to sensitive kinetic studies of RdRps, requiring only 6nM 3D(pol) to obtain a reliable estimate of the initial velocity in as little as 4 min. The assay can immediately accommodate the use of both homopolymer and heteropolymer RNA templates lacking uridylates and can be adapted to RNA templates containing uridine by substituting alpha-thio ATP for ATP. The low background signal produced by other NTPs can be corrected from no enzyme (RdRp) controls. The effect of RdRp/RNA template preincubation was assessed using NS5B and a homopolymer RNA template and a time-dependent increase of RdRp activity was observed. Progress curves for a chain terminator (3(')-deoxyguanosine 5(')-triphosphate) and an allosteric NS5B inhibitor demonstrated the predicted time- and dose-dependent reductions in signal. This assay should facilitate detailed kinetic studies of RdRps and their potential inhibitors using either standard or single-nucleotide approaches.
[Show abstract][Hide abstract] ABSTRACT: Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.
Journal of Virology 02/2004; 78(2):700-9. DOI:10.1128/JVI.78.2.700-709.2004 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A9C9, 16D9A4 and 20A12C7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A9C9, and between 282 and 372 for 16D9A4 and 20A12C7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.
Virus Research 07/2001; 75(2):179-87. DOI:10.1016/S0168-1702(01)00239-8 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Internal ribosomal entry sites (IRESs) can function in foreign viral genomes or in artificial dicistronic mRNAs. We describe an interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5'-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion up to nt 40 in the HCV 5' nontranslated region relieved the replication block, yielding PV/HCV variants replicating to high titers. Fortuitous yet crippling interactions between an IRES and surrounding heterologous RNA must be considered when IRES-based dicistronic expression vectors are being constructed.
Journal of Virology 08/2000; 74(13):6223-6. DOI:10.1128/JVI.74.13.6223-6226.2000 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: H.-H. Lu and E. Wimmer (Proc. Natl. Acad. Sci. USA 93:1412-1417, 1996) have demonstrated that the internal ribosomal entry site (IRES) of poliovirus (PV) can be functionally replaced by the related genetic element from hepatitis C virus (HCV). One important finding of this study was that open reading frame sequences 3' of the initiating AUG, corresponding to the open reading frame of the HCV core polypeptide, are required to create a viable chimeric virus. This made necessary the inclusion of a PV 3C protease (3Cpro) cleavage site for proper polyprotein processing to create the authentic N terminus of the PV capsid precursor. Chimeric PV/HCV (P/H) viruses, however, grew poorly relative to PV. The goal of this study was to determine the molecular basis of impaired replication and enhance the growth properties of this chimeric virus. Genetic modifications leading to a different proteinase (PV 2Apro) cleavage site between the HCV core sequence and the PV polyprotein (P/H701-2A) proved far superior with respect to viral protein expression, core-PV fusion polyprotein processing, plaque phenotype, and viral titer than the original prototype PV/HCV chimera containing the PV 3Cpro-specific cleavage site (P/H701). We have used this new virus model to answer two questions concerning the role of the HCV core protein in P/H chimeric viral proliferation. First, a derivative of P/H701-2A with frameshifts in the core-encoding sequence was used to demonstrate that production of the core protein was not necessary for the translation and replication of the P/H chimera. Second, a viral construct with a C-terminal truncation of 23 amino acids of the core gene was used to show that a signal sequence for signal peptidase processing, when present in the viral construct, is detrimental to P/H virus growth. The novel P/H chimera described here are suitable models for analyzing the function(s) of the HCV elements by genetic analyses in vivo and for antiviral drug discovery.
Journal of Virology 03/1999; 73(2):1546-54. · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma. We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation. Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro. A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized. Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell lines. Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES. In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line. Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.
Journal of Virology 08/1998; 72(7):5638-47. · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2A(pro), 2BC, 2C, and 2B. Specifically, 2A(pro) can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. transdominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome.
Journal of Virology 03/1998; 72(2):1297-307. · 4.44 Impact Factor