Human immunodeficiency virus integration complexes are active following ordered addition of wild type integrase, viral DNA, and LEDGF/p75

Human immunodeficiency virus (HIV-1) requires integration of the viral genome into the host DNA for replication. Efficient HIV-1 integration employs a host co-factor LEDGF/p75 to stabilize the HIV-1 integration complex and tether that complex to host chromatin. Integration may be studied with purified components HIV-1 integrase (IN), LEDGF/p75, and DNA mimicking the ends of the viral DNA genome (vDNA) assembled as an intasome. There is a likely order of addition during infection with HIV-1 IN binding to vDNA before encountering LEDGF/p75. However, the ordered assembly of wild type HIV-1 IN, LEDGF/p75, and oligomer vDNA has not been tested. Variable assemblies occurred on ice before the addition of target DNA. Incubation on ice and addition of LEDGF/p75 were required to assemble complexes capable of efficient concerted integration. Integration efficiency following variable order of addition of intasome components was greatest when LEDGF/p75 was added last to preassembled HIV-1 IN and vDNA.