National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
U54 AI150472
United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
R01 AI136680
United States
Citation
Journal: Nucleic Acids Res / Year: 2022 Title: B-to-A transition in target DNA during retroviral integration. Authors: Ilona K Jóźwik / Wen Li / Da-Wei Zhang / Doris Wong / Julia Grawenhoff / Allison Ballandras-Colas / Sriram Aiyer / Peter Cherepanov / Alan N Engelman / Dmitry Lyumkis / Abstract: Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of ...Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.
Name: ZINC ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: ZN
Molecular weight
Theoretical: 65.409 Da
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Macromolecule #6: CALCIUM ION
Macromolecule
Name: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: CA
Molecular weight
Theoretical: 40.078 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 6.5 Component:
Concentration
Name
Formula
25.0 mM
Tris-HCl
200.0 mM
sodium chloride
NaCl
2.0 mM
DTT
10.0 mM
Calcium chloride
CaCl2
25.0 mM
Zinc chloride
ZnCl2
Grid
Model: UltrAuFoil / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec.
Vitrification
Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C.
Details
MMTV STC intasomes 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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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-100 / Number grids imaged: 1 / Number real images: 1578 / Average exposure time: 10.0 sec. / Average electron dose: 67.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Initial model building was accomplished by rigid-body fitting of the MMTV CSC structure downloaded from the Protein Data Bank (PDB ID: 3JCA) into the EM map in Chimera 1.14 by Fit in Map tool. Unmodeled protein and DNA residues were manually built in Coot 0.9.4.1 and the structure underwent a few iterative cycles of manual model re-building and real-space refinement in Phenix. Ramachandran and secondary structure restraints were applied. To model the full octameric intasome, we additionally rigid-body docked the flanking IN dimers (PDB ID: 5CZ2) into the map. The density connecting the flanking dimers and the core was evident, but broken, and therefore a model was not derived for the linker regions. The final model accounts for the complete octameric MMTV STC intasome with connections for the linker regions.
Refinement
Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: CC
Output model
PDB-7usf: Mouse mammary tumor virus strand transfer complex intasome
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