Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1996-08-23
1998-04-14
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 5, 435 76, 435219, 536 232, 536 2372, C12Q 137, C12Q 170, C12N 950, C07H 2104
Patent
active
057390029
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
This present invention has as its subject a method for reconstituting the serine protease activity associated with the HCV NS3 protein, which makes use of the ability of the HCV protein NS4A, or sequences contained therein, to act as a cofactor of the serine protease activity or more generally speaking of enzymatic activities associated with NS3.
As is known, the hepatitis C virus (HCV) is the main etiological agent of non-A, non-B hepatitis (NANB). It is estimated that HCV causes at least 90% of post-transfusional NANB viral hepatitis and 50% of sporadic NANB hepatitis. Although great progress has been made in the selection of blood donors and in the immunological characterization of blood used for transfusions, there is still a high level of acute HCV infection among those receiving blood transfusions (one million or more infections every year throughout the world). Approximately 50% of HCV-infected individuals develop cirrhosis of the liver within a period that can range from 5 to 40 years. Furthermore, recent clinical studies suggest that there is a correlation between chronic HCV infection and the development of hepatocellular carcinoma.
HCV is an enveloped virus containing an RNA positive genome of approximately 9.4 kb. This virus is a member of the Flaviviridae family, the other members of which are the flaviviruses and the pestiviruses. The RNA genome of HCV has recently been mapped. Comparison of sequences from the HCV genomes isolated in various parts of the world has shown that these sequences can be extremely heterogeneous. The majority of the HCV genome is occupied by an open reading frame (ORF) that can vary between 9030 and 9099 nucleotides. This ORF codes for a single viral polyprotein, the length of which can vary from 3010 to 3033 amino acids. During the viral infection cycle, the polyprotein is proteolytically processed into the individual gene products necessary for replication of the virus. The genes coding for HCV structural proteins are located at the 5'-end of the ORF, whereas the region coding for the non-structural proteins occupies the rest of the ORF.
The structural proteins consists of C (core, 21 kDa), E1 (envelope, gp.sup.37) and E2 (NS1, gp61). C is a non-glycosylated protein of 21 kDa which probably forms the viral nucleocapsid. The protein E1 is a glycoprotein of approximately 37 kDa and it is believed to be a structural protein for the outer viral envelop. E2, another membrane glycoprotein of 61 kDa, is probably a second structural protein in the outer envelope of the virus.
The non-structural region starts with NS2 (p24), a hydrophobic protein of 24 kDa whose function is unknown. NS2. A protein of 68 kDa which follows NS2 in the polyprotein, is predicted to have two functional domains: a serine protease domain in the first 200 amino-terminal amino acids, and an RNA-dependent ATPase domain at the carboxy terminus. The gene region corresponding to NS4 codes for NS4A (p6) and NS4B (p26), two hydrophobic proteins of 6 and 26 kDa, respectively, whose functions have not yet been clarified. The gene corresponding to NS5 also codes for two proteins, NS5A (p56) and NS5B (p65), of 56 and 65 kDa, respectively. In amino acid sequence present in all the RNA-dependent RNA polymarases can be recognized within the NS5 regions. This suggests that the NS5 region contains parts of the viral replication machinery.
Various molecular biological studies indicate that the signal peptidase, a protease associated with the reticulum of the host cell, is responsible for proteolytic processing in the non-structural region, that is to say at sites C/E1, E1/E2 and E2/NS2. The serine protease contained in NS3 is responsible for cleavage at the junctions between NS3 and NS4A, between NS4A and NS4B, between NS4B and NS5A and between NS5A and NS5B. In particular it has been found that the cleavage performed by this serine protease leaves a residue of cysteine or threonine on the amino-terminal side (position P1) and an alanine or serine residue on the carboxy-terminal side (position P1') of t
REFERENCES:
Grakoui et al. (1993) J. Virology 67, No.5, 2832-2843.
Journal of Virology, vol. 68, No. 6, 1994, pp. 3753-3760. C. Fatilla et a "Both NS3 and NS4 are required for Proteolytic processing of HCV nonstructural proteins."
Journal of Virology, vol. 67, No. 7, 1993, pp. 4017-4026. L. Tomei et al., "NS3 is a serine protease required for processing of HCV polyprotein."
Virology, vol. 204, 1994, pp. 163-169. S.S. Leinbach et al., "Substrate specificity of the NS3 proteinase of HCV . . . ".
Proc. Natl. Acad. Sci. USA, vol. 91, 1994, pp. 888-892. E. Pizzi et al., "Molecular model of the specificity pocket of the HCV protease . . . ".
De Francesco Raffaele
Failla Christina
Tomei Licia
Istituto di Richerche di Biologia Molecolare P. Angeletti S.p.A.
Slobodyansky Elizabeth
Wax Robert A.
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