National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
U54 AI150472
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
R01 AI136680
米国
The Francis Crick Institute
FC10061
英国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
P50AI150481
米国
引用
ジャーナル: Nat Commun / 年: 2022 タイトル: Multivalent interactions essential for lentiviral integrase function. 著者: 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 / ...著者: 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 / 要旨: 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- ...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. 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.
凍結剤: ETHANE / 装置: HOMEMADE PLUNGER 詳細: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C.
詳細
MVV CSC intasomes, assembled and purified as previously described, were applied onto R1.2/1.3 gold UltrAufoil grids, Au 300 mesh (Quantifoil). Cryo-EM grids were prepared by manually freezing using a manual plunger in cold room at 4C and stored in liquid nitrogen for future data acquisition.
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電子顕微鏡法
顕微鏡
FEI TALOS ARCTICA
詳細
The stage was tilted to 40 degrees during data collection to account for the preferential orientation of the sample within the vitreous ice.
The STC model refined in study, with the tDNA removed, was docked into the CSC cryoEM map using UCSF Chimera. It was observed that there were some slight differences in some domain positions, to address this, individual domains that were not well fitted to the map were docked as individual domains to achieve a best-fit starting model. Two (C2 related) NTDs (aa 1-35) were removed from the model due to lack of supporting map. Adjustments were made to the model interactively using Coot and the coordinates were subjected to real-space refinement in Phenix dev-4213-000 employing C2 NCS constraints.
精密化
空間: REAL / プロトコル: FLEXIBLE FIT / 温度因子: 262 / 当てはまり具合の基準: CC
得られたモデル
PDB-7u32: MVV cleaved synaptic complex (CSC) intasome at 3.4 A resolution