Cloning and Biochemical Characterization of HIV Integrase
Abstract
Our long range goal is to understand the macromolecular interactions responsible for the integration of HIV DNA into human chromosomes. The primary goal of this project is to develop appropriate biochemical assays for the interaction of the HIV integration protein, IN, with a specific DNA target, the viral LTRs. The gene encoding IN has been subcloned from infectious viral DNA into appropriate cloning vectors and has been expressed in both E. coli and in insect cells. IN has been expressed as a pol fusion protein that is processed by HIV protease in vivo and as a single protein carrying an added methionine residue. The lack of any additional carboxy-terminal processing of IN has been demonstrated. Purification of the recombinant IN is in progress. Potential DNA substrates for IN have been constructed. These include a synthetic oligonucleotide substrate corresponding to the LTR end and a substrate containing the ligated junction of the two LTRs, obtained by polymerase chain amplification from HIV-infected cells. The latter provides both linear and circular DNA forms. During the construction of the circle junction clone, it was found that 50% of the circle junction sequences in infected cells are aberrant, containing deletions or insertions at the circle junction. The involvement of IN in the production of these forms will be investigated. Keywords: RA 1; Viral DNA; Viral endonuclease; Retrovirus; Lentivirus; Genome; Molecular virology.
Document Details
- Document Type
- Technical Report
- Publication Date
- Dec 15, 1989
- Accession Number
- ADA217468
Entities
People
- Ellen Murphy
Organizations
- Public Health Research Institute