Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs.
HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors.
Alian
Akram
A
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA.
Griner
Sarah L
SL
Chiang
Vicki
V
Tsiang
Manuel
M
Jones
Gregg
G
Birkus
Gabriel
G
Geleziunas
Romas
R
Leavitt
Andrew D
AD
Stroud
Robert M
RM
D002384:Catalysis; D004279:DNA, Viral / Q000378:metabolism*; D004220:Disulfides; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000493:pharmacokinetics*; D015497:HIV-1 / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization
D004279:DNA, Viral; D004220:Disulfides; D019428:HIV Integrase Inhibitors; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1