HIV-1 Integrase Preassembled on Donor DNA Is Refractory to Activity Stimulation by LEDGF/p75

Abstract

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.

Identification and Characterization of PWWP Domain Residues Critical for LEDGF/p75 Chromatin Binding and Human Immunodeficiency Virus Type 1 Infectivity

“…Purified LEDGF/p75 protein potently stimulates the activities of recombinant lentiviral integrase proteins (4,6,7,62), and the nature of the in vitro reaction conditions can influence the requirements for the different host factor functions. Direct binding to HIV-1 integrase, for example, is crucial under all conditions (6,8,45,48,62,71), whereas N-terminal deletion mutants of LEDGF/p75 defective for chromatin binding retained about half the level of wild-type activity when integration was performed with naked target DNA (1, 62). Recombinant ⌬PWWP/MutL1 protein by contrast functioned at only ca.…”

Affinities between the Binding Partners of the HIV-1 Integrase Dimer-Lens Epithelium-derived Growth Factor (IN Dimer-LEDGF) Complex

“…Construction of LEDGF Expression Vectors-Isolation of the LEDGF cDNA and the construction of pLEDGF-6His, a plasmid expressing a C-terminally His 6 -tagged LEDGF protein, have been described previously (25). All subsequent LEDGF variants were created using pLEDGF-6His as the starting plasmid.…”

Integrase, LEDGF/p75 and HIV replication

“…The interaction between p75 and IN is direct [73] and it is confined to only one of the seven retroviral genera, lentivirinae [75,87,100]. A number of laboratories established the role of p75 in chromatin tethering, mapped relevant functional domains, and worked to answer the question of virological relevance [74][75][76][77][78][79][80][81][82][85][86][87][88][89][90][91]93,[95][96][97][100][101][102][103][104][105][108][109][110]. The first indication of the protein's significance was the clarification it provided about the vexing issue of the intracellular trafficking of retroviral integrase proteins.…”