Intergrase assay

• US 6,727,058 B2
• Filed: 03/12/2001
• Issued: 04/27/2004
• Est. Priority Date: 03/18/1998
• Status: Expired due to Fees

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Hansen, Mark, Bushman, Frederic
• Primary Examiner(s): Stucker, Jeffrey
• Assistant Examiner(s): Hill, Myron G
• Application Number:
  US09/803,863
• Publication Number:
  US 20010009772A1
• Time in Patent Office: 1,142 Days
• Field of Search: 435/5, 435/6, 435/69.1, 435/239, 435/183
• US Class Current: 435/5
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/16052 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 2740/16122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2740/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2760/20222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The present invention provides packaging cell lines and recombinant lentiviral or retroviral particles produced therefrom, particularly pseudotyped retroviral particles. The packaging cell lines of the invention are produced by inducibly expressing an envelope protein by methods described herein. Also described is a screening assay for compounds that affect integration of viral nucleic acid into target (e.g., host) nucleic acid. Such compounds are identified based on their effect on viral integrase.

Claims

1. A method of detecting a viral nucleic acid sequence integrated into a target nucleic acid sequence comprising:

a) immobilizing a target nucleic acid sequence to a solid support;

b) contacting the target nucleic acid with a viral preintegration complex (PIC) containing a viral nucleic acid sequence, under conditions sufficient to allow the viral nucleic acid sequence to integrate into the target nucleic acid sequence;

c) releasing the immobilized target nucleic acid sequence; and

d) detecting the integrated viral nucleic acid sequence in the released target nucleic acid sequence.

2. The method of claim 1, wherein the viral preintegration complex (PIC) is isolated from HIV-infected cells.

3. The method of claim 1, wherein integration of the viral nucleic acid sequence in the target nucleic acid is detected using a polymerase chain reaction.

4. The method of claim 1, wherein integration of the viral nucleic acid sequence in the target nucleic acid is detected by nucleic acid probe hybridization under stringent conditions.

5. The method of claim 1, wherein the viral preintegration complex (PIC) is isolated from HIV vector infected cells.

6. The method of claim 1, further comprising quantifying the viral nucleic acid sequence in the released target nucleic acid sequence.

7. A method of identifying a compound that modulates viral integrase activity contained in a preintegration complex (PIC) comprising:

a) immobilizing a target nucleic acid sequence to a solid support;

b) contacting the target nucleic acid of a) with a viral preintegration complex (PIC) containing a viral nucleic acid sequence, under such conditions and for sufficient time so as to allow the viral nucleic acid sequence to integrate into the target nucleic acid sequence;

c) contacting the preintegration complex with a compound suspected of modulating integrase activity prior to, or simultaneously with, b);

d) releasing the immobilized target nucleic acid sequence; and

e) detecting the integrated viral nucleic acid sequence;

wherein the amount of viral nucleic acid sequence integrated in the released target nucleic acid sequence is indicative of the effect of the compound on integrase activity.

8. The method of claim 7, wherein the effect of the compound on integrase activity is inhibition.

9. The method of claim 7, wherein the viral integrase activity is HIV integrase.

10. The method of claim 7, wherein integration of the viral nucleic acid sequence in the target nucleic acid is detected using a polymerase chain reaction.

11. The method of claim 7, wherein integration of the viral nucleic acid sequence in the target nucleic acid is detected by nucleic acid probe hybridization under stringent conditions.

12. The method of claim 7, wherein the viral preintegration complex (PIC) is isolated from HIV vector infected cells.

13. The method of claim 7, further comprising quantifying the viral nucleic acid sequence in the released target nucleic acid sequence.