Multivalent interactions essential for lentiviral integrase function

Allison Ballandras-Colas; Vidya Chivukula; Dominika T. Gruszka; Zelin Shan; Parmit K. Singh; Valerie E. Pye; Rebecca K. McLean; Gregory J. Bedwell; Wen Li; Andrea Nans; Nicola J. Cook; Hind J. Fadel; Eric M. Poeschla; David J. Griffiths; Javier Vargas; Ian A. Taylor; Dmitry Lyumkis; Hasan Yardimci; Alan N. Engelman; Peter Cherepanov

doi:10.1038/s41467-022-29928-8

Multivalent interactions essential for lentiviral integrase function

Allison Ballandras-Colas, Vidya Chivukula, Dominika T. Gruszka, Zelin Shan, Parmit K. Singh, Valerie E. Pye, Rebecca K. McLean, Gregory J. Bedwell, Wen Li, Andrea Nans, Nicola J. Cook, Hind J. Fadel, Eric M. Poeschla, David J. Griffiths, Javier Vargas, Ian A. Taylor, Dmitry Lyumkis, Hasan Yardimci, Alan N. Engelman, Peter Cherepanov

Research output: Contribution to journal › Article › peer-review

Abstract

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 subunits¹. 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 Å 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 α-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.

Original language	English (US)
Article number	2416
Journal	Nature communications
Volume	13
Issue number	1
DOIs	https://doi.org/10.1038/s41467-022-29928-8
State	Published - Dec 2022

ASJC Scopus subject areas

Chemistry(all)
Biochemistry, Genetics and Molecular Biology(all)
Physics and Astronomy(all)

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10.1038/s41467-022-29928-8

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Ballandras-Colas, A., Chivukula, V., Gruszka, D. T., Shan, Z., Singh, P. K., Pye, V. E., McLean, R. K., Bedwell, G. J., Li, W., Nans, A., Cook, N. J., Fadel, H. J., Poeschla, E. M., Griffiths, D. J., Vargas, J., Taylor, I. A., Lyumkis, D., Yardimci, H., Engelman, A. N., & Cherepanov, P. (2022). Multivalent interactions essential for lentiviral integrase function. Nature communications, 13(1), [2416]. https://doi.org/10.1038/s41467-022-29928-8

Ballandras-Colas, A, Chivukula, V, Gruszka, DT, Shan, Z, Singh, PK, Pye, VE, McLean, RK, Bedwell, GJ, Li, W, Nans, A, Cook, NJ, Fadel, HJ, Poeschla, EM, Griffiths, DJ, Vargas, J, Taylor, IA, Lyumkis, D, Yardimci, H, Engelman, AN & Cherepanov, P 2022, 'Multivalent interactions essential for lentiviral integrase function', Nature communications, vol. 13, no. 1, 2416. https://doi.org/10.1038/s41467-022-29928-8

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title = "Multivalent interactions essential for lentiviral integrase function",

abstract = "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 subunits1. 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 {\AA} 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 α-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.",

author = "Allison Ballandras-Colas and Vidya Chivukula and Gruszka, {Dominika T.} and Zelin Shan and Singh, {Parmit K.} and Pye, {Valerie E.} and McLean, {Rebecca K.} and Bedwell, {Gregory J.} and Wen Li and Andrea Nans and Cook, {Nicola J.} and Fadel, {Hind J.} and Poeschla, {Eric M.} and Griffiths, {David J.} and Javier Vargas and Taylor, {Ian A.} and Dmitry Lyumkis and Hasan Yardimci and Engelman, {Alan N.} and Peter Cherepanov",

note = "Funding Information: We thank Massimo Palmarini for CPT3 cells, Didier Trono for a generous gift of pMD2.G, Ron Vale for His-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). 10 Publisher Copyright: {\textcopyright} 2022, The Author(s).",

year = "2022",

month = dec,

doi = "10.1038/s41467-022-29928-8",

language = "English (US)",

volume = "13",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "Nature Publishing Group",

number = "1",

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TY - JOUR

T1 - Multivalent interactions essential for lentiviral integrase function

AU - Ballandras-Colas, Allison

AU - Chivukula, Vidya

AU - Gruszka, Dominika T.

AU - Shan, Zelin

AU - Singh, Parmit K.

AU - Pye, Valerie E.

AU - McLean, Rebecca K.

AU - Bedwell, Gregory J.

AU - Li, Wen

AU - Nans, Andrea

AU - Cook, Nicola J.

AU - Fadel, Hind J.

AU - Poeschla, Eric M.

AU - Griffiths, David J.

AU - Vargas, Javier

AU - Taylor, Ian A.

AU - Lyumkis, Dmitry

AU - Yardimci, Hasan

AU - Engelman, Alan N.

AU - Cherepanov, Peter

N1 - Funding Information: We thank Massimo Palmarini for CPT3 cells, Didier Trono for a generous gift of pMD2.G, Ron Vale for His-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). 10 Publisher Copyright: © 2022, The Author(s).

PY - 2022/12

Y1 - 2022/12

N2 - 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 subunits1. 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 Å 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 α-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.

AB - 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 subunits1. 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 Å 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 α-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.

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DO - 10.1038/s41467-022-29928-8

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VL - 13

JO - Nature Communications

JF - Nature Communications

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ER -

Multivalent interactions essential for lentiviral integrase function

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