- EMDB-5773: A Two-Pronged Structural Analysis of Retroviral Maturation Indica... -
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Basic information
Entry
Database: EMDB / ID: EMD-5773
Title
A Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition
Map data
Reconstruction of RSV-CASP 30 nm outer capsid T=3
Sample
Sample: Icosahedral assembly of Rous sarcoma virus capsid proteins with spacer peptide, 30 nm outer particle
Journal: J Virol / Year: 2013 Title: A two-pronged structural analysis of retroviral maturation indicates that core formation proceeds by a disassembly-reassembly pathway rather than a displacive transition. Authors: Paul W Keller / Rick K Huang / Matthew R England / Kayoko Waki / Naiqian Cheng / J Bernard Heymann / Rebecca C Craven / Eric O Freed / Alasdair C Steven / Abstract: Retrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence ...Retrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence suggests that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persists of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into icosahedral capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation.
History
Deposition
Oct 23, 2013
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Header (metadata) release
Nov 6, 2013
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Map release
Nov 6, 2013
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Update
Dec 4, 2013
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Current status
Dec 4, 2013
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Organism: Escherichia coli (E. coli) / Recombinant strain: BL21
Virus shell
Shell ID: 1 / Name: outer capsid / Diameter: 300 Å / T number (triangulation number): 3
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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
Concentration
2 mg/mL
Buffer
pH: 7.5 Details: 10 mM Tris-HCl, 75 mM sodium chloride, 0.05 mM EDTA, 0.5 M sodium phosphate
Grid
Details: Holey carbon film on R2/2 400 mesh copper grid
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93.15 K / Instrument: LEICA KF80 / Details: Vitrification carried out in nitrogen atmosphere. Method: 4.0 microliter sample dropped onto grid, blotted on one side for 2 second, then plunged.
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Electron microscopy
Microscope
FEI/PHILIPS CM200FEG
Temperature
Average: 93.15 K
Date
Sep 24, 2011
Image recording
Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 17 / Average electron dose: 15 e/Å2 / Details: scanning at 4000 dpi / Bits/pixel: 16
Electron beam
Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
30 nm particles were picked manually, extracted, and CTF corrected by phase reversal. Particle orientation search and refinement were performed using image package Bsoft.
CTF correction
Details: CTF was determined from the whole micrograph. Phase reversal was applied to each particle.
Final reconstruction
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Bsoft / Number images used: 612
FSC plot (resolution estimation)
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