The integration of viral DNA (vDNA) into host chromatin establishes an infection in target cells and is a hallmark of the replication cycle of all retroviruses, including the human immunodeficiency virus. Integration is catalyzed by the intasome, a complex consisting of multimer of viral integrase proteins and the terminal ends of viral DNA (vDNA). Most prior work used free target DNA to probe retroviral integration, largely due to experimental simplicity. However, eukaryotic host target DNA is chromatinized, with nucleosomes forming the core structural building blocks of the genome. Accordingly, our understanding of intasome-mediated integration into chromatin remains incomplete. Here, we used the mouse mammary tumor virus (MMTV) as a model to study chromatin integration. We employed nucleosome reconstitution and sequencing strategies, biochemical binding and activity assays, and high-resolution cryogenic electron microscopy (cryo-EM) to mechanistically interrogate how and where MMTV integrates its vDNA into chromatin substrates. We solved multiple intasome-nucleosome complexes to reveal that MMTV selectively and simultaneously engages across two nucleosomes when transferring the viral genome into the host chromatin. Based on these structures, we mutagenized specific amino acid residues on the interface between intasomes and the substrate DNA, which we are currently testing enzymatically and through viral infections to define the functional outcome of abrogating cross-nucleosome interactions. Our hypothesis is that cross-nucleosome interactions guide the MMTV intasome toward more compact, and thus transcriptionally silent, regions of chromatin. Collectively, these studies will advance mechanistic studies underlying the key integration step catalyzed by retroviruses. Our work will also have important implications for understanding how proteins engage across extended stretches of chromatin to mediate functional outcomes.
Gamez et al. (Sun,) studied this question.