Multivalent interactions essential for lentiviral integrase function

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The authors determined high-resolution cryo-EM structures of the lentiviral intasome - the nucleoprotein complex that inserts viral DNA into a host chromosome - and show that the architecture comprising 16 integrase subunits is critical for its function. A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits(1). Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 angstrom resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the alpha-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.
© The Author(s) 2022. Artículo escrito por 20 autores. We thank Massimo Palmarini for CPT3 cells, Didier Trono for a generous gift of pMD2.G, Ron Vale for His10-PS-SNAPf vector, Goedele Maertens for sharing the luciferase assay protocol and critical reading of the manuscript, Massimo Pizzato for advice on RT assays and nucleofection, P. Walker and A. Purkiss for computer and software support, and M. Singer for help with tissue culture. This work was funded by US National Institutes of Health grants P50 AI150481 (P.C. and A.N.E.), R01 AI070042 (A.N.E.), and U54 AI150472 (D.L.); US National Science Foundation CAREER MCB-2048095, the Margaret T. Morris Foundation, and the Hearst Foundations (D.L.); the Spanish Ministry of Science and Innovation PID2019-108850RA-I00 (JV); and the Francis Crick Institute (P.C., H.Y., and I.A.T.), which receives its core funding from Cancer Research UK (FC001061, FC001221, FC001178), the UK Medical Research Council (FC001061, FC001221, FC001178), and the Wellcome Trust (FC001061, FC001221, and FC001178).