Claims
- 1. A surrogate cell-based reporter system to evaluate the protease activity of Hepatitis C virus NS3 protease comprising a mammalian cell transfected with:
a) a first chimeric DNA molecule comprising:
i) a non-cytopathic expression system for inducing expression of said first chimera upon transfection in said mammalian cell; ii) an HCV recombinant DNA molecule operably linked to said expression system, said HCV DNA molecule encoding an NS3-5 polyprotein comprising:
an active NS3 protease, an NS4A protein sufficient to allow embedding in the ER membrane upon translation and acting as co-factor for the active NS3 protease activity, NS4B and NS5A proteins of sufficient length to allow correct structural orientation of the active NS3 protease relative to an NS5A/5B target cleavage site, and NS5B protein or a fragment thereof sufficient to provide said NS5A/5B cleavage site for said active NS3 protease; and iii) a transactivator domain fused downstream of said HCV DNA molecule, said transactivator domain encoding a transactivator molecule capable of initiating expression of a reporter gene; and b) a second chimeric DNA molecule encoding said reporter gene co-joined to an operon responding to said transactivator molecule; wherein expression of said first recombinant molecule leads to the production of a fusion polyprotein anchored to the endoplasmic reticulum of said mammalian cell, said anchored protein being cleaved by said active NS3 protease thereby allowing translocation of said transactivator domain for inducing expression of said reporter gene as a means to evaluate said active NS3 protease activity.
- 2. The surrogate cell-based reporter system according to claim 1, wherein said NS3-5 polyprotein comprises a NS3 domain, variants or fragments thereof sufficient to provide an active protease once translated.
- 3. The surrogate cell-based reporter system according to claim 1, wherein said NS3 protein is truncated to exclude helicase domain or a NS3 protein where the helicase domain is inactivated.
- 6. The surrogate cell-based reporter system according to claim 1, wherein said HCV recombinant DNA molecule comprises a nucleotide sequence coding for the full length NS3 polyprotein of HCV.
- 8. The reporter system according to claim 2, wherein said first chimeric DNA molecule is as defined in SEQ ID NO. 1.
- 9. The reporter system according to claim 2, wherein said first chimeric DNA molecule encodes a polyprotein of SEQ ID NO. 2.
- 10. The surrogate cell-based reporter system according to claim 1, wherein said first chimeric DNA molecule encodes the precursor polyprotein comprising all of the functional cleavage sites of: NS3/4A, 4A/4B, 4B/5A, and 5A/5B.
- 11. The surrogate cell-based reporter system according to claim 1, wherein said NS5A/5B cleavage site is cleavable by said active NS3 protease whereas one or more of NS3/4A; 4A/4B; or 4B/5A cleavage site is a variant not cleavable by said active NS3 protease.
- 12. The surrogate cell-based reporter system according to claim 1, wherein said non-cytopathic promoter system is selected from the group consisting of: the CMV promoter, the SV40 early-promoter system, and the RSV (Rous Sarcoma virus) LTR promoter system.
- 14. The surrogate cell-based reporter system according to claim 1, wherein said transactivator is selected from the group consisting of: tetracycline transactivator (tTA), NFκB, HIV-1 tat and GAL-4.
- 15. The surrogate cell-based reporter system according to claim 14, wherein said transactivator is tTA.
- 16. The surrogate cell-based reporter system according to claim 1, wherein said reporter gene encodes a molecule selected from the group consisting of: secreted alkaline phosphatase, β-galactosidase, luciferase, chloramphenicol aminotransferase and green fluorescent protein.
- 17. The surrogate cell-based reporter system according to claim 16, wherein said reporter molecule is SEAP or luciferase.
- 18. The surrogate cell-based reporter system according to claim 17, wherein said reporter molecule is SEAP.
- 19. The surrogate cell-based reporter system according to claim 1, wherein said transactivator is tTA and said reporter is luciferase or SEAP.
- 20. The surrogate cell-based reporter system according to claim 19, wherein said transactivator is tTA and said reporter is SEAP.
- 22. The surrogate cell-based reporter system according to claim 1, wherein said transactivator domain is located at the 3′-end of the first chimeric DNA molecule.
RELATED APPLICATIONS
[0001] This application is a divisional of prior U.S. application Ser. No. 09/563,899 filed May 3, 2000, which claims, as does the present application priority to U.S. provisional application No. 60/132,360 filed May 4, 1999, the disclosures of all of which are incorporated by reference in their entirety.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60132360 |
May 1999 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09563899 |
May 2000 |
US |
Child |
10328127 |
Dec 2002 |
US |