Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-05-11
2001-11-27
Houtteman, Scott W. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S235100
Reexamination Certificate
active
06322966
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to identification of optimal properties of an RNA template for “copy-back” self-priming RNA synthesis of hepatitis C virus replicase. This activity can be used to screen for anti-HCV replicase compounds, or to characterize the biological relevance of lead compounds that have already been identified.
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)].
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., Science 258: 135-140 (1992); Kao et al., J. Med. Virol. 50:303-308 (1996); Shimizu et al., J. Virol. 68:1494-1500 (1994); Wyatt et al., J. Virol. 72:1725-1730 (1998)]. Development of small molecule inhibitors directed against specific viral targets has thus become the focus of anti-HCV research. The determination of crystal structures for NS3 protease [Kim et al., Cell 87:343-355 (1996); Love et al., Cell 87:331-342 (1996); Yan et al., Protein Sci. 7:837-847 (1998)] and NS3 RNA helicase [Yao et al., Nat. Struct. Biol. 4:463-467 (1997)] has provided important structural insights for rational design of specific inhibitors.
One key enzyme encoded by HCV is NS5B, which has been shown to be an RNA-dependent RNA polymerase (RdRp) [Al et al., Virus Res. 53: 141-149 (1998); Behreus et al., EMBO J. 15:12-22 (1996); DeFrancesco et al., Methods Enzymol. 275:58-67 (1996); Lohmann et al., J. Virol 71:8416-8428 (1997); Yuan et al., 1997, Biochem. Biophys. Res. Commun. 232:231-235 (1997); Ferrari et al., J. Virol. 73:1649-54 (1999)]. NS5B is thus believed to be responsible for HCV genome replication. Cellular localization studies revealed that NS5B is membrane associated and distributed in the perinuclear region [Hwang et al., Annu. Rev. Biochem. 63:777-822 (1997)]. This coincides with the distribution of NS5A [Tanji et al., J. Virol. 69:1575-1581(1995)], suggesting that NS5A and NS5B may stay together after proteolytic cleavage at the NS5A/NS5B junction. It has been postulated that the nonstructural proteins of HCV (NS3 to NS5B) may assemble into membrane-associated replication complexes that are competent for authentic RNA genome replication.
By itself, HCV NS5B RdRp appears to lack specificity for HCV RNA and can “copy back” heterologous nonviral RNA. This lack of specificity for HCV RNA may reflect the notion that additional viral or cellular factors are required for specific recognition of the replication signal, most likely present at the 3′ untranslated region.
The ability of recombinant HCV NS5B to initiate RNA synthesis by a primer-independent mechanism has recently been observed [U.S. patent application Ser. No. 09/305,185 filed May 4, 1999, entitled “De Novo Priming Activity of Hepatitis C Virus Replicase,”. De novo priming was also observed with bovine viral diarrhea virus [Kao et al., Virology, 23, pp. 1-7(1999)]. De novo initiation of RNA synthesis is likely to be the mode of initiation of HCV replication in an infected cell.
Although de novo priming/initiation is likely to be the mode of RNA replication employed by HCV, the recombinant polymerase alone can replicate the entire HCV RNA genome initiated from an intramolecular 3′ stemloop (via a copy-back primer), resulting in a near dimer-size RNA product (Lohmann et al., J. Virol. 71:8416-28 (1997); Lohmann et al., Virol. 249:108-118 (1998)].
Thus, there is a need in the art to resolve the current uncertainty concerning HCV genome replication.
There is a further need in the art to develop effective therapeutic strategies to inhibit viral specific features of HCV replicase.
The present invention addresses these and other needs in the art.
SUMMARY OF THE INVENTION
The present invention advantageously establishes the RNA template characteristics that optimize the elongative RNA synthesis of hepatitis C virus (HCV) replicase via a copy-back mechanism. The features that have been identified can be harnessed for various primary, and especially secondary, screening assays for anti-HCV compounds. In particular, the present invention permits one to determine whether an HCV NS5B binding compound or inhibitor is capable of selectively blocking HCV NS5B replicase activity, without adversely affecting host cell replication and transcription processes.
Thus, in one embodiment, the invention provides an assay system for hepatitis C virus (HCV) replicase activity. The assay system comprises an RNA template that has an unstable, small stemloop at the 3′ end and is capable of supporting copy-back RNA synthesis. Also provided in the assay system are an enzymatically active amount of HCV non-structural protein 5B (NS5B), ATP, GTP, CTP, and UTP nucleoside triphosphates (NTPs), wherein one of the NTPs is radiolabeled, and an assay buffer that supports replication activity of NS5B.
The invention also provides a method for detecting hepatitis C virus (HCV) replicase activity. This method measures the nucleic acid synthesized by an HCV non-structural protein 5B (NS5B) on an RNA template that has an unstable, small stemloop at the 3′ end and is capable of forming a copy-back structure in the presence of ATP, GTP, CTP, and UTP nucleoside triphosphates (NTPs), wherein one of the NTPs is radiolabeled, and an assay buffer that supports replication activity of NS5B.
Thus, one object of the invention is to provide an assay system for characterizing the HCV replicase activity via copy-back mechanism.
Another object of the invention is to provide a method for mechanistic and kinetic analysis of HCV replicase.
Another object of the invention is to provide ch
Hong Zhi
Lau Johnson Y. N.
Zhong Weidong
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