HIV-1 integrase preassembled on donor DNA is refractory to activity stimulation by LEDGF/p75.
LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step. In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at the start of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.
Yu
Fang
F
Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, USA.
Jones
Gregg S
GS
Hung
Magdeleine
M
Wagner
Alice H
AH
MacArthur
Holly L
HL
Liu
Xiaohong
X
Leavitt
Stephanie
S
McDermott
Martin J
MJ
Tsiang
Manuel
M
D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D046911:Macromolecular Substances / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; C119380:lens epithelium-derived growth factor; D004247:DNA; D019427:HIV Integrase