Correct integration mediated by integrase-LexA fusion proteins incorporated into HIV-1.
Fusion of wild-type or truncated integrase to a sequence-specific DNA-binding protein, such as the Escherichia coli LexA repressor, results in an integration bias toward the recognition site of the DNA-binding protein in vitro. Integrase-defective HIV-1 could become integration-competent by supplying the fusion protein in trans. To understand the mechanism of complementation, the virus-host DNA junctions of cells infected with the integrase-LexA containing virus were sequenced. The characteristic hallmarks of wild-type integration were present, a 5'-TG/CA-3' at the ends of the viral sequence and a 5-bp direct repeat in the immediately flanking cellular DNA. Experiments were also carried out to determine the mechanism by which the amino- or carboxy-terminal truncated integrase fused to LexA restored integration to the integrase-mutant viral clone. Complementation experiments using purified fusion proteins in vitro, or viruses encoding a C-terminal truncated integrase and containing various fusion proteins in trans, indicated that the truncated integrase-LexA proteins are inactive per se and they restore integration by forming mixed multimers with the virally encoded mutant integrase. Correct integration of retroviral DNA by the in trans method illustrates the feasibility of introducing integrase fusion proteins into retroviral vectors to achieve site-directed integration without interfering with the attributes of the integration reaction.
Holmes-Son
Michelle L
ML
Department of Molecular and Medical Pharmacology, UCLA AIDS Institute, Los Angeles, California, 90095, USA.
Chow
Samson A
SA
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018389:Codon, Nonsense; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011533:Proviruses / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism; D016662:Virus Integration; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D018389:Codon, Nonsense; D029968:Escherichia coli Proteins; D019426:Integrases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein