National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)
Intramural Research Program
米国
引用
ジャーナル: mBio / 年: 2021 タイトル: Cryo-Electron Tomography of the Herpesvirus Procapsid Reveals Interactions of the Portal with the Scaffold and a Shift on Maturation. 著者: Michael H C Buch / William W Newcomb / Dennis C Winkler / Alasdair C Steven / J Bernard Heymann / 要旨: Herpes simplex virus 1 (HSV-1) requires seven proteins to package its genome through a vertex in its capsid, one of which is the portal protein, pUL6. The portal protein is also thought to facilitate ...Herpes simplex virus 1 (HSV-1) requires seven proteins to package its genome through a vertex in its capsid, one of which is the portal protein, pUL6. The portal protein is also thought to facilitate assembly of the procapsid. While the portal has been visualized in mature capsids, we aimed to elucidate its role in the assembly and maturation of procapsids using cryo-electron tomography (cryoET). We identified the portal vertex in individual procapsids, calculated a subtomogram average, and compared that with the portal vertex in empty mature capsids (A-capsids). The resulting maps show the portal on the interior surface with its narrower end facing outwards, while maintaining close contact with the capsid shell. In the procapsid, the portal is embedded in the underlying scaffold, suggesting that assembly involves a portal-scaffold complex. During maturation, the capsid shell angularizes with a corresponding outward movement of the vertices. We found that in A-capsids, the portal translocates outward further than the adjacent capsomers and strengthens its contacts with the capsid shell. Our methodology also allowed us to determine the number of portal vertices in each capsid, with most having one per capsid, but some none or two, and rarely three. The predominance of a single portal per capsid supports facilitation of the assembly of the procapsid. Herpes simplex virus 1 (HSV-1) infects a majority of humans, causing mostly mild disease but in some cases progressing toward life-threatening encephalitis. Understanding the life cycle of the virus is important to devise countermeasures. Production of the virion starts with the assembly of an icosahedral procapsid, which includes DNA packaging proteins at a vertex, one of which is the dodecameric portal protein. The procapsid then undergoes maturation and DNA packaging through the portal, driven by a terminase complex. We used cryo-electron tomography to visualize the portal in procapsids and compare them to mature empty capsids. We found the portal located inside one vertex interacting with the scaffold protein in the procapsid. On maturation, the scaffold is cleaved and dissociates, the capsid angularizes, and the portal moves outward, interacting closely with the capsid shell. These transformations may provide a basis for the development of drugs to prevent HSV-1 infections.
名称: Human alphaherpesvirus 1 / タイプ: virus / ID: 1 / 親要素: 0 / 含まれる分子: #1 詳細: Procapsids were obtained from infected Vero cells. Purification steps were carried out at room temperature. Harvested cells were resuspended in PBS buffer and lysed by addition of TX-100. ...詳細: Procapsids were obtained from infected Vero cells. Purification steps were carried out at room temperature. Harvested cells were resuspended in PBS buffer and lysed by addition of TX-100. Procapsids were purified by multiple filtration steps using Eppendorf spin filters. Turquoise pellets containing procapsids were resuspended in PBS, sonicated in a bath sonicator, and concentrated to the desired volume using a 300 kDa MWCO filter. NCBI-ID: 10298 / 生物種: Human alphaherpesvirus 1 / Sci species strain: KOS / ウイルスタイプ: VIRUS-LIKE PARTICLE / ウイルス・単離状態: SPECIES / ウイルス・エンベロープ: Yes / ウイルス・中空状態: No
モデル: Quantifoil / 材質: COPPER / メッシュ: 300 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY / 前処理 - タイプ: PLASMA CLEANING / 前処理 - 雰囲気: OTHER 詳細: Grids were plasma-cleaned in a Model 1020 plasma cleaner (Fischione, Export, PA).
凍結
凍結剤: ETHANE / チャンバー内湿度: 90 % / 装置: LEICA EM GP 詳細: Sample was mixed with Aurion Anionic 10 nm BSA gold tracer (EMS) at a 1:1 ratio, and 3 uL were applied to Quantifoil copper grids covered with 300-mesh holey carbon (EMS). Excess liquid was ...詳細: Sample was mixed with Aurion Anionic 10 nm BSA gold tracer (EMS) at a 1:1 ratio, and 3 uL were applied to Quantifoil copper grids covered with 300-mesh holey carbon (EMS). Excess liquid was blotted off for 2.5 s, and grids were flash-frozen in liquid ethane..
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
撮影
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / デジタル化 - 画像ごとのフレーム数: 1-2 / 撮影したグリッド数: 1 / 平均露光時間: 1.0 sec. / 平均電子線量: 3.1 e/Å2 詳細: Tilts were imaged in movie mode with 2 movies at 0.5 seconds each.