- EMDB-5774: A Two-Pronged Structural Analysis of Retroviral Maturation Indica... -
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基本情報
登録情報
データベース: EMDB / ID: EMD-5774
タイトル
A Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition
マップデータ
Reconstruction of the inner particles (T=1) of the 30 nm RSV-CASP
試料
試料: Icosahedral assembly of Rous sarcoma virus capsid proteins with spacer peptide (inner particle)
ジャーナル: J Virol / 年: 2013 タイトル: A two-pronged structural analysis of retroviral maturation indicates that core formation proceeds by a disassembly-reassembly pathway rather than a displacive transition. 著者: 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 / 要旨: 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.
pH: 7.5 詳細: 10 mM Tris-HCl, 75 mM sodium chloride, 0.05 mM EDTA, 0.5 M sodium phosphate
グリッド
詳細: Holey carbon film on R2/2 400 mesh copper grid
凍結
凍結剤: ETHANE / チャンバー内湿度: 90 % / チャンバー内温度: 93.15 K / 装置: LEICA KF80 / 詳細: Vitrification carried out in nitrogen atmosphere. 手法: 4.0 microliter sample dropped onto grid, blotted on one side for 2 second, then plunged.
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電子顕微鏡法
顕微鏡
FEI/PHILIPS CM200FEG
温度
平均: 93.15 K
日付
2011年9月24日
撮影
カテゴリ: FILM / フィルム・検出器のモデル: KODAK SO-163 FILM デジタル化 - スキャナー: NIKON SUPER COOLSCAN 9000 デジタル化 - サンプリング間隔: 6.35 µm / 実像数: 17 / 平均電子線量: 15 e/Å2 / 詳細: scanning at 4000 dpi / ビット/ピクセル: 16
30 nm particles were picked manually, extracted, and CTF corrected by phase reversal. Particle orientation search (using ~20 nm icosahedral sphere as the initial reference) and refinement were performed using image package Bsoft.
CTF補正
詳細: CTF was determined from the whole micrograph. Phase reversal was applied to each particle.
最終 再構成
アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 20.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: Bsoft 詳細: A ~20 nm icosahedral sphere was used for initial orientation search. 使用した粒子像数: 612