Core nucleosomes are refractory to lentiviral DNA integration

HIV-1 and other lentiviruses hijack the cellular chromatin-binding protein LEDGF/p75 to facilitate integration into active transcription units. However, the mechanism of chromatin engagement by lentiviral intasomes and the structural role of LEDGF/p75 in this process remain poorly understood. To address these gaps, we studied the activities of native HIV-1 preintegration complexes and in vitro-assembled lentiviral intasomes in the presence of chromatinized target DNA. While LEDGF/p75 was both essential and minimally sufficient to enhance lentiviral integration into chromatin containing histone H3 trimethylated on Lys36, it unexpectedly facilitated integration outside of the nucleosome core particles. LEDGF/p75 additionally inhibited integration into unmodified chromatin in a dose-dependent manner, promoting integration into naked DNA. To explore the structural foundation for these activities, we imaged maedi-visna virus intasomes saturated with LEDGF/p75 before and after strand transfer by cryogenic electron microscopy. The structures revealed that the host factor alters the target DNA binding platform of the lentiviral intasome, imposing significant constraints on the path and configuration of target DNA to impede nucleosome engagement. Our results establish the preference of lentiviral intasomes for linker DNA regions within H3K36Me3-enriched chromatin and show that LEDGF/p75 plays a specific structural role at the viral-host target DNA interface.