Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-06-25
2001-12-04
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S023000, C435S069700, C435S070100, C435S091330, C435S235100, C435S325000, C536S023400, C536S023720
Reexamination Certificate
active
06326137
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a chimeric pestivirus, such as bovine viral diarrhea virus (BVDV), that depends on a hepatitis C virus (HCV) protease function. The invention further relates to using the chimeric, infectious virus to screen for HCV NS3 inhibitor compounds in cell culture systems or in animal models of viral infection.
BACKGROUND OF THE INVENTION
Infection by hepatitis C virus (HCV) is a compelling human medical problem. HCV is recognized as the causative agent for most cases of non-A and non-B hepatitis, with an estimated prevalence of 170 million cases (i.e., 2-3%) globally [Choo, et al., Science, 244: 359-362 (1989); Kuo, et al., Science, 244: 362-364 (1989); Purcell, FEMS Microbiology Reviews, 14: 181-192 (1994); Van der Poel, C. L., Current Studies in Hematology and Blood Transfusion, H. W. Reesink, Ed., (Basel: Karger), pp. 137-163 (1994)]. Four million individuals may be infected in the United States alone [Alter, and Mast, Gastroenterol. Clin. North Am., 23: 437-455 (1994)].
Upon first exposure to HCV only about 10% or less of infected individuals develop acute clinical hepatitis, while others appear to resolve the infection spontaneously. In the most instances, however, the virus establishes a chronic infection that persists for decades [Iwarson, FEMS Microbiology Reviews, 14: 201-204 (1994)]. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma [Kew, FEM Microbiology Reviews, 14: 211-220 (1994); Saito, et al., Proc. Natl. Acad. Sci. USA 87:6547-6549 (1990)]. Currently, there are no broadly effective treatments for the debilitating progression of chronic HCV.
HCV is an enveloped positive-stranded RNA virus beloning to the Flaviviridae family. The HCV genome encodes a polyprotein of 3010-3033 amino acids [Choo, et al. Proc. Natl. Acad. Sci. USA, 88: 2451-2455 (1991); Kato, et al., Proc. Natl. Acad. Sci. USA, 87: 9524-9528 (1990); Takamizawa, et al., J. Virol., 65: 1105-113 (1991)]. The HCV nonstructural (NS) proteins provide catalytic machinery for viral replication. The NS proteins are derived by proteolytic cleavage of the polyprotein [Bartenschlager, et al., J. Virol., 67: 3835-3844 (1993); Grakoui, et al. J. Virol, 67: 2832-2843 (1993); Grakoui, et al., J. Virol., 67: 1385-1395 (1993); Tomei, et al., J. Virol., 67: 4017-4026 (1993)].
The order and nomenclature of the cleavage products are as follows: NH2-C-E1-E2-p7-NS2-NS4A-NS3-NS4B-NS5A-NS5B-COOH (
FIG. 1
) [Grakoui et al., 1993, J. Virol. 67:1385-95; Hijikata et al., 1991, PNAS 88:5547-51; Lin et al., 1994, J. Virol. 68:5063-73]. The three amino-terminal putative structural proteins, C (capsid), E1, and E2 (two envelope glycoproteins), are believed to be cleaved by a host signal peptidase of the endoplasmic reticulum (ER). The host enzyme is also responsible for generating the amino terminus of NS2. The proteolytic processing of the nonstructural proteins are carried out by the viral proteases: NS2-3 and NS3, contained within the viral polyprotein. The NS2-3 protease catalyzes the cleavage between NS2 and NS3. It is a metalloprotease and requires both NS2 and the protease domain of NS3.
The NS3 protease catalyzes the rest of the cleavages in the nonstructural part of the polyprotein. The NS3 protein contains 631 amino acid residues and is comprised of two enzymatic activities: the protease domain contained within amino acid residues 1-181 and a helicase ATPase domain contained within the rest of the protein [Kim et al., 1995, Biochem Biophys Res. Comm., 215:160-166]. It is not known if the 70 kD NS3 protein is cleaved further in infected cells to separate the protease domain from the helicase domain, although no cleavage has been observed in cell culture expression studies.
The NS3 protease is a member of the serine protease family. Its active site consists of a His, Asp, Ser catalytic triad. Mutation of the Ser residue abolishes the ability to cleave at NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions. The cleavage between NS3 and NS4A is intramolecular, whereas the cleavages at the NS 4A/4B, 4B/5A, 5A/5B sites may occur in trans.
Experiments using transient expression of various forms of HCV NS polyproteins in mammalian cells have established that the NS3 serine protease is necessary but not sufficient for efficient processing of all of these cleavages. HCV NS3 protease requires a cofactor to catalyze some of these cleavage reactions. Efficient proteolytic processing at NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites within the non-structural domain of hepatitis C virus requires a heterodimeric complex of the NS3 serine protease and the NS4A protein. [Bartenschlager et al. 1995, J. Virol. 67:3835-3844; Failla et al., 1994, J. Virol. 68:3753-3760]. A 13-amino acid synthetic NS4A peptide, corresponding to the central hydrophobic domain of NS4A protein, spanning residues 21-33 has been shown to be sufficient for activation of NS3 protease [Butkiewicz et al., 1996, Virology, 225: 328-338]. A smaller domain (amino acid residues 22-30) of NS4A has been shown to be sufficient for activation of the protease [Lin et al., 1995, J. Virol 69:4377-80].
The recently published three dimensional structure of the NS3 protease [im et al, 1996, Cell 87:343-355; Yan et al, 1998, Protein Sci. 7:837-847] revealed that the N-terminal 37 residues of NS3 adopt a &bgr; (residues 6-9)-&agr; (residues 14-22)-&bgr; (residues 33-37) structure upon binding of a synthetic peptide corresponding to the central hydrophobic domain spanning residues 21-32 of NS4A protein.
The Flaviviridae family comprises two other genera of single-stranded positive-sense RNA viruses: flaviviruses and pestiviruses [Rice, In
Virology, Third Ed,
B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Raven Press: New York (1996)]. Bovine viral diarrhea virus (BVDV) is a prototype virus in the Pestivirus genus. Its RNA genome is one of the largest (12.5 kb) among members of the Flaviviridae family [Collett, et al., Virol. 165:191-199 (1988)]. As one of the most characterized members of the Flaviviridae family, BVDV provides a good model system for HCV. Similar to HCV, it consists of (1) a long 5′ untranslated region (UTR), which contains an internal ribosomal entry site (IRES) for translation of viral proteins [Poole, et al., Virol. 206:750-754 (1995); Frolov, et al., RNA 4:1418-1435 (1998); Chon, et al., Virol. 251:370-381 (1998)]; and (2) a single large open reading frame (ORF) which encodes a polyprotein of approximately 3,900 amino acids [Meyers and Thiel., Adv. Virus Res. 47:53-118 (1996); Collett, supra; Rice, supra]. Unique to pestiviruses, the first virally encoded protein is Npro, a cysteine protease, only responsible for the cleavage between Npro and the capsid protein (C) [Stark, et al., J. Viro. 67:7088-7095 (1993); Rumenapf, et al., J. Virol. 72:2544-2547 (1998)]. Interestingly, Npro is not essential for viral growth and its coding region can be replaced by a ubiquitin gene directly fused in-frame to the capsid gene. The resulting chimeric virus has growth properties similar to the wild type viruses [Tratschin, et al., J. Virol. 72:7681-7684 (1998)]. Several other investigators have also demonstrated that Npro is not required for pestiviral replication [Mittelholzer, et al., Virus Res. 51:125-137 (1997); Behrens, et al., J. Virol. 72:2364-2372 (1998)].
Current therapies with alpha interferon alone and the combination of alpha interferon-ribavirin have been shown to be effective in a portion of patients with chronic HCV infection [Marcellin et al., Ann. Intern. Med. 127:875-881 (1997); Reichard et al., Lancet 351:83-87 (1998)]. Vaccine development has been hampered by the high degree of immune evasion and the lack of protection against reinfection, even with the same inoculum [Farci et al., Scie
Hong Zhi
Lai Vicki C. H.
Lau Johnson Y. N.
Schering Corporation
Schram David B.
Wortman Donna C.
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