The Hepatitis C virus (HCV) is the major etiologic agent for non-A, non-B hepatitis. It is estimated that around 400 million people or >2% of the world's population are infected (Di Bisceglie, A. M. (1998) Hepatitis C. Lancet 351:351-5; Houghton M. (1996), p. 1035-1058. In B. N. Fields and D. M. Knipe and H. P. M. (ed.), Fields' Virology, 3rd ed. Lippincott-Raven, Philadelphia/New York). HCV usually results in a chronic infection in 60 to 80% of infected individuals with 20% having progression to cirrhosis, hepatocellular carcinoma or chronic liver failure. At least 6 major viral genotypes and over 50 proposed subtypes of HCV have been identified worldwide (Simmonds, P. et al. (1994) Identification of genotypes of hepatitis C virus by sequence comparisons in the core, E1 and NS-5 regions. J Gen Virol 75:1053-1061). These genotypes are based on nucleotide and amino acid sequence diversity with the most divergent isolates differing by more than 30% (Bréchot, C. (1996) Hepatitis C virus: Molecular biology and genetic variability. Digestive Diseases and Sciences 41:6S-21S; Bukh, J. et al. (1995) Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Semin Liver Dis 15:41-63). Among the different types, there are regions which are highly conserved and have sequence homology close to 100%, however, in highly variable regions such as the envelope proteins homology is <70% (Booth, J. C. et al. (1998) Comparison of the rate of sequence variation in the hypervariable region of E2/NS1 region of hepatitis C virus in normal and hypogammaglobulinemic patients. Hepatology 27:223-27; Hayashi, N. et al. (1993) Molecular cloning and heterogeneity of the human hepatitis C virus (HCV) genome. J Hepatol 17:S94-107; Hijikata, M. et al. (1991) Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun 175:220-228; Kato, N. et al. (1992) Marked sequence diversity in the putative envelope proteins of hepatitis C viruses. Virus Res 22:107-123; Lesniewski, R. R. et al. (1993) Hypervariable 5′-terminus of hepatitis C virus E2/NS1 encodes antigenically distinct variants. J Med Virol 40:150-156; Pozzetto, B. et al. (1996) Structure, genomic organization, replication and variability of hepatitis C virus. Nephrol Dial Transplant 11 [Suppl 4]:2-5; Vizmanos, J. L. et al. (1998) Degree and distribution of variability in the 5′ untranslated, E1, E2/NS1 and NS5 regions of the hepatitis C virus (HCV). J Viral Hepat 5:227-240).
HCV is a member of the Flaviviridae family whose other members include the Flaviviruses and Pestiviruses (Kato, N. et al. (1991) Molecular structure of the Japanese hepatitis C viral genome. FEBS Lett 280:325-328). The HCV genome is a positive-stranded RNA of ˜9.5 kb which contains a single open reading frame encoding a polyprotein of 3010 to 3033 amino acids (Takamizawa, A. et al. (1991) Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol 65:1105-1113). Proteolytic processing of the polyprotein is accomplished by host and viral proteases. Host signal peptidases cleave the structural proteins which are located in the 5′ end, while two viral proteases cleave the non-structural proteins. These cleavages result in at least 10 viral proteins in the order NH2—C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. There are also non-coding regions located at the 5′ and 3′ ends which are involved in ribosome binding and replication initiation, respectively.
One of the viral proteases, the NS3 protease, is encoded by the N-terminal region of the HCV NS3 gene. It consists of 181 amino acids and is a chymotrypsin-like serine-protease responsible for cleavage of the non-structural proteins of HCV (Bartenschlager, R. et al. (1993) Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 67:3835-3844; Eckart, M. R. et al. (1993) The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor. Biochem Biophys Res Commun 192:399-406; Gallinari, P. et al. (1998) Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus. J Virol 72:6758-6769; Hahm, B. et al. (1995) NS3-4A of hepatitis C virus is a chymotrypsin-like protease. J Virol 69:2534-2539; Hijikata, M. et al. (1993) Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 67:4665-4675; Hijikata, M. et al. (1993) Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci USA. 90:10773-10777; Manabe, S. et al. (1994) Production of nonstructural proteins of hepatitis C virus requires a putative viral protease encoded by NS3. Virology 198:636-644; Tomei, L. et al. (1993) NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol 67:4017-4026). Processing of the structural proteins by NS3 occurs in a well ordered cascade with the first cleavage occurring between NS3 and NS4A followed by NS5A-NS5B, NS4A-NS4B and NS4B-NS5A (Bartenschlager, R. et al. (1994) Kinetic and structural analysis of hepatitis C virus polyprotein processing. J Virol 68:5045-5055; D'Souza, E. D. et al. (1994) Analysis of NS3-mediated processing of the hepatitis C virus non-structural region in vitro. J Gen Virol 75:3469-3476; Eckart M. R., et al., 1993, supra; Failla, C. et al. (1994) Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol 68:3753-3760; Kolykhalov, A. A. et al. (1994) Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing. J Virol 68:7525-7533; Shimotohno, K. et al. (1995) Processing of the hepatitis C virus precursor protein. J Hepatol 22:87-92; Shoji, I. et al. 1999 Internal processing of hepatitis C virus NS3 protein. Virology 254:315-323; Tomei, L. et al., 1993, supra). The cleavage between NS3 and NS4A occurs in cis, while the other cleavages are in trans. The virus-encoded cofactor, NS4A is necessary for efficient NS3 function. Proteolytic processing efficiency has been shown to increase dramatically in the presence of the NS4A protein (Failla, C. et al. (1995) An amino-terminal domain of the hepatitis C virus NS3 proteinase is essential for the interaction with NS4A. J Virol 69:1769-1777; Failla, 1994, supra; Gallinari, P. et al. 1999 Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A. Biochemistry 38:5620-5632; Lin, C. et al. (1995) A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro. J Virol 69:4373-4380; Satoh, S. et al. (1995) The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A. J Virol 69:4255-4260; Tanji, Y. et al. (1995) Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing. J Virol 69:1575-1581). In addition, NS3 contains a tetrahedrally bound zinc atom, which appears to play a structural role (De Francesco, R. et al. (1996) A zinc binding site in viral serine proteinases. Biochemistry 35:13282-13287; Stempniak, M. et al. (1997) The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme. J Virol 71:2881-2886).
Recently, considerable progress has been made in determining how the NS3 protease processes the HCV polypeptide (Kolykhalov, A. A. et al., 1994 supra; Steinkühler, C. et al. (1996) Activity of purified hepatitis C virus protease NS3 on peptide substrates. J Virol 70:6694-6700; Urbani, A. et al. (1997) Substrate specificity of the hepatitis C virus serine protease NS3. J Biol Chem 272:9204-9209). Models for how the protease interacts with cofactors and the substrate have identified four domains, which are involved in enzyme function Barbato, G. et al. 1999. The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism. J Mol Biol 289:371-384). These are the catalytic triad, cofactor and metal binding sites and the substrate-binding pocket. These domains are well defined and contain amino acid residues that are highly conserved in all HCV protease genes sequenced to date (See FIG. 1 in Holland-Staley, C. A., et al. (2002) Genetic diversity and response to IFN of the NS3 protease gene from clinical strains of the hepatitis C virus. Arch Virol 147:1385-1406, which is incorporated herein by reference). Despite this recent burst of structural information and studies showing direct involvement and conservation of amino acid residues, the impact of natural sequence variability on enzyme function is not well understood. In addition, the effects of anti-viral therapy on the NS3 protease sequence are unknown. Elucidation of the natural genetic diversity of the HCV NS3 protease in patient samples is of significant medical as well as theoretical interest. Though all HCV NS3 proteases sequenced to date contain conserved active-site amino acids, sequence variation throughout the NS3 gene is significant (Martell, M. et al. (1992) Hepatitis C virus (HCV) circulates as a population of different but closely related genomes:quasispecies nature of HCV distribution. J Virol. 66:3225-32; Okamoto, H. et al. (1992) Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee: variability and stability. Virology 190:894-899; Okamoto, H. et al. (1992) supra). Also, the presence of multiple species within the same patient, known as quasispecies, creates a potential problem for drug development and resistance. Understanding the extent of NS3 sequence variation in clinical strains will allow more effective development of drugs targeting the HCV protease. For this, data must be available that describes sequence variability as it occurs in HCV-infected persons.
Helicases are enzymes that are responsible for unwinding DNA/DNA, RNA/DNA and RNA/RNA duplexes in a 3′-to-5′ direction. (Bartenschlager, R. et al., 1993, supra; Hahm B., et al., 1995, supra; Tomei, et al., 1993, supra). In addition, helicase enzymes have been proposed to play roles in viral replication and recombination, viral control of host cellular functions, mRNA stability including splicing or processing, transcription, transport, and translation initiation of RNA (Lüking, et al. (1998) The protein family of RNA helicases. Crit. Rev. Biochem. Mol. Biol. 33:259-296). The HCV NS3 helicase/NTPase contains 450 amino acids. The NTPase component hydrolyzes nucleoside 5′-triphosphates, providing the energy requirements. The helicase/NTPase, along with the NS3 protease and NS5b RNA-dependent polymerase, are believed to make up a large complex, which is responsible for viral RNA replication. Recently, considerable progress has been made in determining the structure of the HCV NS3 helicase and its mechanism of duplex unwinding. At least three different crystal structures have been published (Paolini, Cho et al., 1998, supra; Kim et al., 1998, supra; and Yao et al. 1997, supra). In all three, the enzyme was shown to contain 3 domains, with domains 1 and 2 being structurally similar. The enzyme contains seven motifs, including two motifs which are involved in NTP-binding and hydrolysis (motif I [GxGKS] and motif II [DExH]) and one that is involved in ATP hydrolysis and RNA unwinding (motif VI). Most of the motifs are located between the structural domains 1 and 2, with domain 3 separated from the other two by the binding of the nucleotide. Investigators have identified highly conserved residues within each motif. These motifs, along with comparison analysis to other helicases, reveal it to be a member of the DEAD-box family of RNA helicases, specifically the DExH subfamily. Magnesium is required for both the helicase and NTPase activities.
The only FDA approved therapy for hepatitis C infection is interferon (IFN) or pegylated-interferon with or without ribaviron. Interferon production has been shown to be induced after infections with bacteria, parasites and viruses as well as in response to tumors. Interferons are secreted proteins in the cytokine family, which indirectly inhibit the viral life cycle by binding to cellular receptors, thus inducing protein synthesis (Hijikata, M. et al. (1993) Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci USA. 90:10773-10777). Interferon inducible genes contain a promoter region, termed the IFN-stimulated response element (ISRE). Over 30 genes are induced by interferon, however, the function of most of these genes is unknown (Holmes, E. C. et al. (2000) The causes and consequences of genetic variation in dengue virus. Trends Microbiol 8:74-77).
Recently it has been suggested that a short stretch of 40 amino acids in the HCV NS5A gene play a role in IFN resistance, however, while this appears true for some Japanese isolates other investigators have conflicting data. The effects of interferon therapy on the other structural genes is unknown.