Project 3. Integration

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Project 3 – Integration Retroviral replication requires integration of reverse-transcribed viral DNA into a host cell chromosome. This process is catalyzed by integrase (IN) in the context of the stable nucleoprotein complex, containing a multimer of IN assembled on viral DNA ends and termed the intasome. Characterization of in vitro-assembled intasomes from many retroviral species elucidated conserved as well as genus-specific features. Lentiviral intasomes harbor large IN homo-oligomers, containing as many as 16 subunits. However, the intasome represents only a small part of the pre-integration complex (PIC) that assembles and mediates intracellular trafficking of the intasome during virus infection. Furthermore, it is currently unclear how the HIV-1 PIC interfaces with its target, chromatinized host cell genomic DNA. This project aims to characterize the structure and properties of the HIV/lentiviral integration machinery using complementary top-down and bottom-up approaches. From the top- down, we will leverage several novel affinity reagents to enrich for PICs from cellular extracts and then study hemi-purified native HIV-1 PICs using advanced DNA footprinting and microscopy techniques. Experiments using HIV-1 PICs will reveal the function of the IN-binding partner LEDGF/p75 and its cognate epigenetic modification (H3K36me3) in integration in the context of chromatin. With the bottom-up approach, we will take advantage of the intasome from maedi-visna virus (MVV, an ovine lentivirus), which, unlike HIV/SIV intasomes, can be assembled in vitro as monodispersed preparations. This 0.6-MDa complex will allow us to characterize the dynamics and the structure of the complex between the lentiviral intasome and chromatin at high temporal and spatial resolutions. The proposed studies will shed light on the architecture of the lentiviral DNA integration apparatus and how it interfaces with host factors and chromosomes, while avoiding suicidal autointegration. Given that IN inhibitors are used worldwide to treat people living with HIV-1, our results will provide unprecedented details of the biological machine that is the target of these highly successful medicines and could reveal new ways to attack the PIC for therapeutic intervention.