Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1995-09-15
2001-02-27
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S015000, C435S183000, C435S193000
Reexamination Certificate
active
06194140
ABSTRACT:
TECHNICAL FIELD
This invention relates to the molecular biology and virology of the hepatitis C virus (HCV). More specifically, this invention relates to (1) carboxy terminus fragments of the HCV NS3 protein having helicase activity and improved solubility in extraction and assay buffers, (2) methods of expressing the novel NS3 protein fragments having helicase activity and improved solubility, (3) recombinant NS3 protein fragments having helicase activity and improved solubility; (4) NS3 protein mutant fragments; and (5) method of using the HCV NS3 protein fragments for screening helicase inhibitors as potential therapeutic agents.
BACKGROUND OF THE INVENTION
Non-A, Non-B hepatitis (NANBH) is a transmissible disease (or family of diseases) that is believed to be virally induced, and is distinguishable from other forms of virus-associated liver disease, such as those caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Epidemiologic evidence suggests that there may be three types of NANBH: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acquired) type. However, the number of causative agents is unknown. Recently, however, a new viral species, hepatitis C virus (HCV) has been identified as the primary (if not only) cause of blood-associated NANBH (BB-NANBH). See for example, PCT WO89/046699; U.S. patent application Ser. No. 7/456,637, filed Dec. 21, 1989; and U.S. patent application Ser. No. 7/456,637, filed Dec. 21, 1989, incorporated herein by reference. Hepatitis C appears to be the major form of transfusion-associated hepatitis in a number of countries, including the United States and Japan. There is also evidence implicating HCV in induction of hepatocellular carcinoma. Thus, a need exists for an effective method for treating HCV infection: currently, there is none.
HCV is a positive strand RNA virus. Upon infection, its genomic RNA produces a large polyprotein that is processed by viral and cellular proteins into at least 10 different viral proteins. Like other positive strand RNA viruses, replication of the positive strand involves initial synthesis of a negative strand RNA. This negative strand RNA, which is a replication intermediate, serves as a template for the production of progeny genomic RNA. This process is believed to be carried out by two or more viral encoded enzymes, including RNA-dependent RNA polymerase and RNA helicase. RNA polymerase copies template RNA for the production of progeny RNA. This enzyme does not synthesize RNA molecules from DNA template.
The RNA helicase unwinds the secondary structure present in the single-strand RNA molecule. The helicase also unwinds the duplex RNA into single-strand forms. Genomic HCV RNA molecules contain extensive secondary structure. Replication intermediates of HCV RNA are believed to be present as duplex RNA consisting of positive and negative strand RNA molecules. The activity of RNA helicase is believed to be crucial to RNA dependent RNA polymerase which requires unwound single stranded RNA molecules as a template. Therefor, the biological activity of helicase is believed to be required for HCV replication.
NS3 proteins of the three genera of the Flaviviridae family: flavivirus, pestivirus and HCV, have been shown to have conserved sequence motifs of a serine-type proteinase and of a nucleoside triphosphatase (NTPase)/RNA helicase. One third of the N′-terminal of the HCV NS3 protein has been shown to be a trypsin like serine proteinase which cleaves the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions. Faila et al.,
J. Virol.
68:3753-3760 (1994). Two thirds of the NS3 C′-terminal fragment has been shown to encode NTPase/RNA helicase activity. Choo et al.,
PNAS,
88:2451-2455 (1991) and Gorbalenya et al.,
Nucleic Acids Res.,
17:4713-4729 (1989). Suzich et al. showed that two thirds of the carboxy terminal fragment of HCV NS3 expressed in
E. coli
had polynucleotide-stimulated NTPase activity.
J. Virol,
67:6152-6158 (1993). Gwack et al., in “NTPase Activity of Hepatitis C Virus NS3 Protein Expressed in Insect Cells”
Mol. Cells.
5(2): 171-175 (1995), showed two HCV NS3 proteins, p70 and p43, were expressed in a baculovirus expression system. The p70 showed a specific NTPase activity that was inhibited by NS3 monoclonal antibodies. Warrener et al., “Pestivirus NS3 (p80) Protein Possesses RNA Helicase Activity,”
J. Virol.
69:1720-1726 (1995), demonstrated that bovine viral diarrhea virus (BVDV) NS3 protein expressed in a baculovirus expression system had a RNA helicase activity. JP 0631 9583A describes the preparation of a helicase protein encoded by HCV by introducing a HCV helicase gene into the non-essential region of a baculovirus. The helicase amino acid sequence is reported as 1200 through 1500 of the HCV polyprotein. All documents mentioned above are incorporated herein in their entirety by reference.
DISCLOSURE OF THE INVENTION
We have now invented recombinant HCV NS3 protein fragments having helicase activity and improved solubility, fusion HCV NS3 protein fragments having helicase activity and improved solubility, truncated and altered HCV NS3 protein fragments having helicase activity and improved solubility, and cloning and expression vectors therefore, and methods for using these protein fragments in screening assays to assess whether a compound is capable of inhibiting RNA helicase activity and thus inhibiting HCV replication.
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Faila et al.,J. Virol.68:3753-3760 (1994).
Choo et al.,PNAS,88:2451-2455 (1991).
Gorbalenya et al.,Nucleic Acids Res.,17:4713-4729 (1989).
Suzich et al.,J. Virol,67:6152-6158 (1993).
Gwack et al.,Mol. Cells.5(2): 171-175 (1995).
Warrener et al., “Pestivirus NS3 (p80) Protein Possesses RNA Helicase Activity,”J. Virol.69:1720-1726 (1995).
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Clewell et al,Proc Nat Acad Sci USA(1969) 62:1159.
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Patent Abstracts of Japan, vol. 095, No. 002, Mar. 31, 1995 & JP 06 319583 (Souyaku Gijutsu Kenkyusho: KK), Nov. 22, 1994.
Jinn et al. (Jul. 20, 1995) Use of &bgr;-N,N-bis[Carboxymethyl]lysine-Modified Peroxidase in Immunoassays. Analytical Biochemistry, vol. 229, pp. 54-60.
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Kim, et al., Biochemical & Biophysical Research Comm., (1995) 215 (No. 1):160-166.
Choe Joonho
Choo Qui-Lim
Han Jang
Houghton Michael
Blackburn Robert P.
Chiron Corporation
Harbin Alisa A.
Slobodyansky Elizabeth
Trujillo Doreen Y.
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