Name: in vitro disassembly of influenza a virus capsids by gradient centrifugation
Uploaded: 2016-03-27T15:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

私たちは、試薬をご提供するため洋平山内とロベルタマンシーニに感謝します。 AHの研究室は、マリー・キュリー初期研修ネットワーク（ITN）、欧州研​​究会議（ERC）によると、スイス国立科学財団（Sinergia）によってサポートされていました。

緩衝液および原液の調製<ol><li> 80ミリリットルののddH 2 Oで390ミリグラムMES水和物および580mgのNaClを、470mgのトリス塩酸塩を溶解することによりMNT緩衝液（20mMのMES、30mMのトリスおよび100mMのNaCl）を準備pH7.4に緩衝液を調整し、のddH 2 Oで最終容量100mlにそれを持って来ます</li><li> 80ミリリットルのdH 2 O毎に9.76グラムのMES水和物を溶解することによって500mMのMES緩衝液の3つの同一の溶液を調製します。濃NaOHで滴定することにより、それぞれ、pHが5.8、5.4にバッファを調整し、5.0。 dH 2 Oで最終容量100mlに各バッファを持参</li><li> 80ミリリットルのdH 2 Oそれぞれに7.88グラムのトリス塩酸塩を溶解することにより、500 mMトリス緩衝液の2つの同一のソリューションを用意し、濃HClで滴定することにより、それぞれ、7.4および6.4にpHを調整します。 dH 2 Oで最終容量100mlにバッファを持参</li><li> dH 2 O中の50％グリセロール溶液を準備し、glycまでよくかき混ぜエロールは均一に混合されます。ガラス瓶にSteritopフィルタユニット（0.22μmの孔サイズ）を通して溶液をフィルタリングします。 </li><li> 10％NP-40ソリューションとのdH 2 O中1MのNaClを準備</li><li> ddH 2 O 4ミリリットル中に2個の錠剤を溶解することにより25倍濃縮されたプロテアーゼ阻害剤を準備</li></ol>グリセロール勾配の調製<ol><li> 5ミリリットルの洗剤バッファマスターミックス準備（pHは7.4、6.4、5.8、5.4、および5.0）のテストに各pH条件の溶液（300mMのNaCl、2％NP-40を、2倍は、プロテアーゼ阻害剤を濃縮します）。この目的のために、1MのNaClの9ミリリットル、10％NP-40の6ミリリットル、25×プロテアーゼ阻害剤の株式の2.4ミリリットル、及び9ミリリットルののddH 2 0を混ぜます。 </li><li>各pH条件ピペット用の50mlコニカルチューブにマスターミックス4.4 mlです。 </li><li> 5.8以上のpH値についてチューブにそれぞれのpH調整500 mMトリスストック溶液の0.6ミリリットルを追加します。 pHが5.8と低いために、それぞれのpH調整の500mM MESストック溶液の0.6ミリリットルを追加します。 </li><li> 5を作ります300mMのNaClを含む緩衝液、2×プロテアーゼ阻害剤、pHが7.4とのddH 2 Oに調整し、60mMのトリスミリリットルこれは、界面活性剤を含まない対照勾配バッファとして機能します。 </li><li>必要に応じて、それぞれ、濃HCl又はNaOHソリューションを追加することにより、pHが7.4、6.4、5.8、5.4、および5.0に溶液のpHを微調整します。 </li><li> 6別の25％グリセロール溶液が得られる界面活性剤含有および界面活性剤を含まない緩衝液の混合物の5ミリリットルに50％のグリセロールストックの5ミリリットルを追加します。 pH指示薬ストリップを用いてpH値を確認します。 注：測定したpHが所望の値と大きく異なる場合は、それぞれの溶液を再度準備する必要があります。 </li><li> 7比：3でのdH 2 Oで50％のグリセロールストックを混合することにより、15％グリセロール溶液を準備します。 </li></ol> IAVの3超遠心<ol><li> 5ミリリットルの注射器や旧姓を使用することにより（13.2ミリリットル、X 89ミリメートル14ミリメートル）の超クリアな遠心チューブに3ミリリットル15％グリセロール溶液を追加DLE（9 cmの長さ21 G、）。これは勾配の整合性を乱す可能性があるとして、管の内壁に水滴を残さないでください。 6遠心分離管、テストに5 pH条件ごとに1つの対照試料のための1つの合計のためにこれを繰り返します。 </li><li>注意深く5mLの注射器と長い針を使用して、15％のグリセロール層の下に3.4 mlの25％グリセロール溶液を置きます。二つの層を混在させないように注意してください。それぞれのpH調整グリセロール溶液でテストするために、すべての6つの条件のためにこれを繰り返します。 注：以下の手順については、クラスII安全キャビネットで作業。 </li><li>静かに1ミリリットルMNT緩衝液で希釈IAV（X31、H3N2）を含む尿膜液を明らかにした30μlのグリセロール勾配を重ねるごとグラデーションの（周り20-30μgの総ウイルスタンパク質のに相当します）。 </li><li>バランスチューブに反対し、SW41超遠心ローターに入れます。 55,000×gで、150分間遠心し、12°C。 </li><li>遠心分離した後、慎重にきれいなパスツールピペットや吸引器を使用して上清を除去し、 すなわち 、両方のグリセロール層。 40μL（1×）非還元LDSサンプル緩衝液中にペレットを再懸濁します。完全にペレットを溶解するには、上下に数回ピペッティングすることが重要です。 1.5ミリリットルのマイクロチューブにサンプルを移します。 </li></ol> 4. SDS-PAGEペレット画分のクマシー染色<ol><li> 10分間95℃ですべてのサンプルを加熱します。これらはSDS-PAGEによって分析されるまで、この時点で、サンプルは-20℃で保存することができます。 </li><li>負荷溶解ペレット20μlのプレキャスト勾配（4から12パーセント）ビス - トリスミニゲル上に、バッファを実行している1×MOPS SDSで200 Vで1時間実行します。 </li><li> ddH 2 O中の40％メタノールおよび10％氷酢酸固定液を作ります</li><li>コロイド状クーマシー液の十分な量で一晩15cmの細胞培養皿に1時間と染色のための固定液中でゲルをインキュベートしながらそっと室温で振とう。 注：これは、染色溶液の蒸発を回避するために、皿を閉鎖することが重要です。 </li><li> ddH 2 O中でゲル脱色ゲルの背景が透明になるまでのddH 2 Oごとに15〜20分を交換してください。それはバンドの定量化のためにスキャンされるまで4℃でのddH 2 Oでゲルを保管してください。 </li><li>高解像度でゲルをスキャンし、タンパク質バンド強度の定量化のために市販されているか、またはカスタムメイドのソフトウェアを使用しています。各バンドに近い領域からバックグラウンド信号を減算し、界面活性剤を含まない対照試料（pH7.4で）にこれらの値を正規化します。 </li></ol>

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The authors have nothing to disclose.

ウイルスキャプシドは、準安定巨大分子複合体です。ビリオンのアセンブリは、ウイルスゲノムのキャプシド形成及び縮合を必要とするが、感染の次のラウンドの開始は、このコンパクトなキャプシド構造の分解に依存します。ウイルスは、細胞受容体、シャペロン、タンパク質分解酵素、モータータンパク質またはヘリカーゼ、ならびにpHおよびイオンスイッチ26,27により提供され?...

インフルエンザAウイルス（IAV）はエンベロープウイルスであり、 オルトミクソウイルス科に属します。その遺伝子は、セグメント化ネガティブセンス一本鎖RNAゲノム上にコードされています。ヒトでは、IAVは、季節の集団発生で発生する呼吸器感染症を引き起こし、世界的なパンデミック1の可能性を負いません。宿主細胞表面2上のシアル酸残基に結合すると、IAVは、クラスリン依存性エンドサイトーシスとクラスリン依存性経路3-8によって内在化されます。エンドサイトーシス液胞における酸性環境（pH値&lt;5.5）は、ウイルスの融合および後期エンドソーム膜9の結果IAVスパイク糖タンパク質ヘマグルチニン（HA）、の主要なコンフォメーション変化を誘発します。サイトO - IAVキャプシドは、（ここではまた、ウイルス「コア」と呼ばれる）後期エンドソーム（LE）をから脱出した後、これを核に、ウイルスリボ（vRNPs）の輸送に続いて細胞質ゾルでコーティングされていませんFウイルスの複製および転写10-13。 HAの酸活性化の前に、ウイルスはその後の解体14-16のためのコアをプライムエンドサイトーシスシステム、内pHの漸減を経験します。この「プライミング」で陽子とK +10,14,16の流入を媒介するウイルス膜中のM2イオンチャネルをステップ。ウイルス内部のイオン濃度の変化は、相互作用がウイルスのマトリックスタンパク質M1と8 vRNPバンドルによって構築の妨げとなり、膜融合10,14-17以下の細胞質にIAVの脱殻を促進します。 プライミング工程の直接的かつ定量的な分析がエンドソームを実験的にアクセスすることが困難であるという事実によって妨げられてきました。エントリー方法は、さらに、非常に非同期です。また、このような放出されたウイルスRNAまたは感染力の測定値の定量RT-PCRのようなエンドポイントアッセイは、ウイルスcapsiの生化学的状態についての詳細な画像を提供していません。エントリの任意の段階で、D。 siRNAまたは薬物治療によってエンドソームの摂動が大幅IAVエントリ18-20の理解に貢献してきましたが、微調整が厳密に制御されたエンドソームの成熟プログラムで非特異的な副作用に困難となりやすいです。 これらの問題を回避するために、我々は速度勾配遠心分離17の使用に基づいて、以前に開発されたin vitroでのプロトコルを適応しています。主にタンパク質分解切断およびEM分析続く界面活性剤処理の組み合わせに基づいていた他の試み21,22、23,24、とは対照的に、このアプローチは、容易に定量化可能な結果を可能にしました。異なる傾斜層を介して遠心分離沈降粒子がに露出されることを可能にし、制御された方法で変化する条件に反応します。提示されたプロトコルでは、IAV清澄尿膜腔液または精製されたウイルス粒子に由来する二層グリセロール中に沈殿さ底部層は、非イオン性界面活性剤NP-40（ 図1）を含有する勾配。ウイルスは二、界面活性剤含有層に入ると、ウ​​イルスの脂質エンベロープおよびエンベロープ糖タンパク質は、静かに可溶化し、残されています。コアは、8 vRNPバンドルで構成され、ペレット画分に安定な構造としてマトリックス層、堆積物に囲まれています。例えばM1およびvRNP関連核タンパク質（NP）、ウイルスコアタンパク質は、SDS-PAGEおよびクマシー染色によってペレットに同定することができます。具体的には、市販の勾配ゲルを利用して高感度コロイド状クーマシー25で染色し、高精度及びウイルスコア関連タンパク質であっても、少量の検出を可能にします。 これは、pH、塩濃度、および推定脱殻因子コア成分及びコアSTABILの沈降挙動に影響を与えるように異なる条件かどうかを試験するための基準を設定します<html>性。この目的のために、グリセロール勾配だけ低く、界面活性剤含有層は、目的の因子または条件を導入することによって改変されます。技術は、IAVコア16の完全に異なるpH値および塩濃度の効果を調査する際に特に貴重でした。 IAVの脱殻の中間ステップは、pHが5.5と低い16,17（ 図1）でvRNP解離に続いて、弱酸性のpH（&lt;6.5）で、マトリクス層の解離を含む監視することができます。後者のステップは、さらに後期エンドサイトーシス環境16を反映し 、グリセロール層中の高K +濃度の存在によって増強されました。したがって、ウイルスおよび勾配を通って沈降コアとして、彼らはそのエンドソームにおける模倣条件変更環境を経験しました。結果は、細胞生物学アッセイから得られた結果を補完し、in vitroでのウイルスコアの段階的分解しました。 tは&quot;&gt;ここで紹介する方法は、高速で有効になっているとIAV（X31およびA / WSN / 33）と、IBV（B /リー/ 40）の再現性の高い分析は、酸性pHによってトリガーされ、K +16,17だけでなく、分解を増加させること脱殻アルカリ性のpH暴露17時のパラミクソウイルスのコアの。アプローチが細胞侵入時にウイルスキャプシド構造およびキャプシド解体の生化学的性質への洞察を得るために、他のエンベロープウイルスに適合させることができると考えられます。

<table><tbody><tr><td>cOmplete&amp;trade;, EDTA-free protease inhibitor tablets</td><td>Sigma-Aldrich</td><td>11873580001</td><td>The stock solution can be stored at 2 to 8 &amp;deg;C for 1 to 2 weeks</td></tr><tr><td>Glacial acetic acid</td><td>Merck Millipore</td><td>100063</td><td></td></tr><tr><td>Glycerol anhydrous BioChemica</td><td>AppliChem</td><td>A1123</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck Millipore</td><td>100317</td><td></td></tr><tr><td>Long injection needle (21&amp;nbsp;G, 9 cm, bevel or blunt-end)</td><td></td><td></td><td></td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>NP-40</td><td>Sigma-Aldrich</td><td>I8896</td><td>Now commercially available as IGEPAL&amp;reg; CA-630</td></tr><tr><td>NuPAGE 4-12% Bis-Tris mini gels, 10&amp;nbsp;wells, 1.0 mm</td><td>Life Technologies</td><td>NP0321</td><td></td></tr><tr><td>NuPAGE LDS sample buffer (4x)</td><td>Life Technologies</td><td>NP0008</td><td></td></tr><tr><td>NuPAGE MOPS SDS running buffer (20x)</td><td>Life Technologies</td><td>NP0001</td><td></td></tr><tr><td>pH indicator strips, pH 4.0-7.0</td><td>Merck Millipore</td><td>109542</td><td></td></tr><tr><td>QC Colloidal Coomassie Stain</td><td>BIO RAD</td><td>1610803</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>106406</td><td></td></tr><tr><td>Sodiumhydroxide</td><td>Merck Millipore</td><td>106498</td><td></td></tr><tr><td>Steritop filter unit</td><td>Merck Millipore</td><td>SCGPT05RE</td><td></td></tr><tr><td>SW41 Ti, ultracentrifuge rotor set</td><td>Beckman Coulter</td><td>331336</td><td></td></tr><tr><td>Thinwall, Ultra-clear centrifuge tubes, 13.2&amp;nbsp;ml, 14 mm x 89 mm</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Tris hydrochloride&amp;nbsp;</td><td>AppliChem</td><td>A1087</td><td></td></tr><tr><td>X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid</td><td>Virapur</td><td></td><td>Freshly thawed on 4 &amp;deg;C</td></tr></tbody></table>

in vitro disassembly of influenza a virus capsids by gradient centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

既に論じたように、エンドソーム内のプライミングコンピ脱殻IAVコアをレンダリングするために必要とされます。コアのプロトンは、（ウイルスRNA、NP、およびポリメラーゼ複雑PB1 / PB2 / PAから成る）M1とvRNPsの相互作用を弱めます。入ってくるウイルスは初期エンドソーム（のEE）6.5（またはそれ以下）のpHに露出してのLEでpH約5.0でのウイルスのヒューズまで継続され...

Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

勾配遠心によるインフルエンザAウイルスキャプシドのインビトロ分解で

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

in-vitro-disassembly-influenza-virus-capsids-gradient

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

8.9K ビュー.  ETH Zurich. Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

ビデオ: In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

ヌクレオカプシドからウイルスDNAの調製

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

免疫・感染学

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

HIV - 1コアのin vitroでの脱殻で

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

シンプルで堅牢な<emin vivoでの>と in vitroで</emウイルスの組み立てを学ぶための>アプローチ

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

感染細胞のクロマチンからのインフルエンザウイルスのリボ核タンパク質複合体のアフィニティ精製

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

インフルエンザウイルスリボ核タンパク質複合体の精製と可視化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

生物学

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

代替体外ウイルスのカプシド構造健全性の定量方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

アセンブリおよびプロトタイプ フォーミー ウイルス Intasomes の精製

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Cell Factory Platformを利用した高力価で高品質なアデノ随伴ウイルスベクターを生成するための合理化および標準化された手順

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

生物工学

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

巨大ウイルス混合物分離のための蛍光活性化細胞選別の代替戦略としての単一細胞マイクロ吸引

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

ダブルヨウ素酸濃度グラジエント遠心分離によるAAV単離

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

著者スポットライト:前臨床応用のための効率的なアデノ随伴ウイルス単離(英語)

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

勾配遠心によるインフルエンザAウイルスキャプシドのインビトロ分解で

要約

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この動画の章

ヌクレオカプシドからウイルスDNAの調製

HIV - 1コアのin vitroでの脱殻で

シンプルで堅牢なと in vitroでアプローチ

感染細胞のクロマチンからのインフルエンザウイルスのリボ核タンパク質複合体のアフィニティ精製

インフルエンザウイルスリボ核タンパク質複合体の精製と可視化

代替体外ウイルスのカプシド構造健全性の定量方法

アセンブリおよびプロトタイプフォーミーウイルス Intasomes の精製

Cell Factory Platformを利用した高力価で高品質なアデノ随伴ウイルスベクターを生成するための合理化および標準化された手順

巨大ウイルス混合物分離のための蛍光活性化細胞選別の代替戦略としての単一細胞マイクロ吸引

In vivoアプリケーションのためのヨジキサノール密度勾配遠心分離によるアデノ随伴ウイルスの浮遊培養産生と精製

勾配遠心によるインフルエンザAウイルスキャプシドのインビトロ分解で

要約

さらに動画を探す

この動画の章

ヌクレオカプシドからウイルスDNAの調製

HIV - 1コアのin vitroでの脱殻で

シンプルで堅牢なと in vitroでアプローチ

感染細胞のクロマチンからのインフルエンザウイルスのリボ核タンパク質複合体のアフィニティ精製

インフルエンザウイルスリボ核タンパク質複合体の精製と可視化

代替体外ウイルスのカプシド構造健全性の定量方法

アセンブリおよびプロトタイプ フォーミー ウイルス Intasomes の精製

Cell Factory Platformを利用した高力価で高品質なアデノ随伴ウイルスベクターを生成するための合理化および標準化された手順

巨大ウイルス混合物分離のための蛍光活性化細胞選別の代替戦略としての単一細胞マイクロ吸引

In vivoアプリケーションのためのヨジキサノール密度勾配遠心分離によるアデノ随伴ウイルスの浮遊培養産生と精製

アセンブリおよびプロトタイプフォーミーウイルス Intasomes の精製