Human immunodeficiency virus type 1 integrase stabilizes a linearized HIV-1 LTR plasmid in vivo.
A mammalian expression vector designed for production of HIV-1 integrase was found to enhance the stability of a linear reporter plasmid in COS-7 cells. The effect is strictly dependent on coexpression of the HIV-1 rev gene and on the inclusion of U3 and U5 portions of the HIV-1 LTR in the reporter plasmid. Integrase point mutations P109S and D116N drastically reduced stabilization whereas T115A and D64A had little or no effect. Immunoblot analysis revealed the presence of a 32-34kDa integrase protein in extracts of transfected COS-7 cells and of wild type and mutant integrase proteins at comparable levels. We conclude that integrase acts in trans in COS-7 cells, possibly by binding to the HIV-1 LTR in the plasmid. This transfection system may be useful for studying factors that stabilize the HIV-1 DNA genome prior to its integration into the host cell chromosome.
Faust
E A
EA
Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, Montreal, Quebec, Canada.
Acel
A
A
Udashkin
B
B
Wainberg
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004254:DNA Nucleotidyltransferases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D015757:Genes, rev; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D019426:Integrases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D016662:Virus Integration
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases