This work was supported by the Academy of Finland (130750 and 218080 to J.T.H.), the Wellcome Trust (090532/Z/09/Z; 089026/Z/09/Z to T.A.B.), and by the MRC (MR/J007897/1 to J.T.H and T.A.B; MR/L009528/1 to T.A.B.).

<ol>
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The authors have nothing to disclose.

<html>ビリオン膜上のウイルス糖タンパク質スパイク構造の知識は、ウイルス複製を理解し、治療し、感染を防ぐために治療薬を開発するために不可欠である。単一粒子の平均と組み合わさ電子低温顕微鏡法は、糖タンパク質スパイクを含むエンベロープウイルス粒子の構造を解決するために最も利用される方法として出現した。しかし、この方法は、対称ウイルスicosahedrallyに制限される。ここでは、Jsubtomoの電子クライオトモグラフィーとsubtomogram平均を適用することにより、私たちは他の現在の構造生物法に適さない多形性エンベロープウイルスに糖タンパク質スパイクを決定するための一般的なプロトコルを概説している。当社の代表的な結果は、この方法の解像度はドメイン構造、オリゴマー化、および無傷のビリオン上の糖タンパク質スパイクのより高次の組織への洞察を明らかにするのに十分であることを示している。 jove_contentは &quot;&gt;このプロトコルの中で最も重要なステップは、互いに統計的に独立した2つの信頼性の出発モデルを構築し、このステップが正常に実行は個人のスパイクができるように、糖タンパク質スパイクが、きつくお互いに詰め十分に大きくしていないことを前提としてい視覚的に認識され、手動で断層像で撮像し、2つの独立したモデルが平均化される。これが不可能な場合は、プロトコルの2つの修正を図ることができる。まず、二つの独立したランダムなモデルが最初にsubtomogramsの2つのランダムなサブセットを定義し、構築することができるこれらのサブセット30内subtomogramsを平均化する。第二 ​​に、孤立したスパイクの構造はX線結晶学により、例えば、他の手段によって導出された場合には、開始モデルとして用いることができるが、注意が低·に注意しなければならない洗練された次のラウンドに2つの結果のモデルが意志としてこのモデルは、低解像度のカットオフ（50〜70オングストローム）を使用して、フィルタを通過これだけの解像度を超えて統計的に独立している。この警告のために、前者のアプローチをお勧めします。 このプロトコルから得られる分解能は四大要因に依存して、i。データ収集の戦略と入力データの品質こと、ii。 subtomograms数、III。アライメントsubtomogramsの正確性、およびiv。構造の不均一性。第一及び第二の制限は、主に高い信号対雑音直接電子CTFと組み合わせた検出器断層撮影法を修正し、自動データ収集を使用することによって克服することができるが、位置合わせ精度がさらに関心自体の構造のサイズおよび形状に影響される。顕著な特徴を欠いている小さなスパイクでこのプロトコルを適用する場合は、アライメント精度、したがって、分解能31を改善するためにスパイクにFabフラグメントを結合することが有利で ​​あり得る。最後に、構造体は、平均化される場合は、出品物の複数の立体配座、サブtomogr午前分類方法は、個別に異なるコンホメーションを平均化するために使用することができる。そのために、Jsubtomoは強力subtomogram分類9を提供し、ディナモ·パッケージに統合されています。 上記のプロトコルは、単離されたウイルス糖タンパク質のX線結晶学に相補的である。結晶構造は、ビリオン膜に対する糖タンパク質の正確な配向を得るために、サブ断層像の平均に嵌合することができる。この方法論の適用は、間違いなくエンベロープウイルスの構造と病理生物学に光を当てるしていきます。

クライオ電子断層撮影法は、複雑な生物学的標本の3次元（3D）再構成の計算を可能にする電子低温顕微鏡イメージング技術である。適当な標本は、全体の原核細胞5および全真核細胞6のさえ薄い領域まで精製高分子複合体1、フィラメント2、被覆小3、および多形性膜ウイルス4の範囲である。傾斜シリーズ、3D断層撮影ボリューム、または断層像のデータ収集に続いて、Bsoft 7およびIMOD 8を含むいくつかの確立されたソフトウェアパッケージを用いて計算することができる。 クライオ電子断層撮影制限対応する断層ボリュームの生物学的解釈による生体試料の研究に固有の二つの側面。まず、有意な放射線損傷を導入する前に、生物学的材料に適用することができる限定された電子線量に起因し、シグナ断層データでlの対雑音比は、典型的には非常に低い。第二に、データ収集中に限られたサンプル傾斜ジオメトリの結果として、オブジェクトの一部のビューは、断層ボリューム内のいわゆる「欠落ウェッジ」アーチファクトにつながる、存在しないままである。断層ボリュームが正常9-12を平均化することができるような巨大分子複合体と同一の構造を、繰り返し含まれている場合は、これらの制限の両方を克服することができる。 断層像再構成の構造を平均する前に、目的のオブジェクトが発見され、同じ方向に揃えておく必要があります。そのような構造体を配置することをしばしば13テンプレートマッチングと呼ばれる手法を用いた断層ボリュームテンプレート構造の相互相関によって達成することができる。このマッチング処理で使用されるテンプレートは、クライオ電子顕微鏡または3D再構成と組み合わさクライオ電子断層撮影法から誘導され得るか、またはそれからシミュレートされた密度マップすることができ原子構造。いくつかの計算のパッケージは、これらのタスク11を実行するために開発されてきた。 例えば、HIV-1のような膜ウイルスの糖タンパク質スパイクの平均化、それらの構造14〜16を研究するための特に成功アプローチしている。構造の理解は、ウイルス - 宿主相互作用の分子基盤の両方を明らかにし、抗ウイルス剤およびワクチン設計の開発をガイドするための不可欠です。巨大分子結晶学は、個別のウイルス糖タンパク質およびそれらの複合体の構造解析（4Åよりも通常より良い）、高解像度のための選択の技術であるが、この方法から得られるX線構造は、ビリオンの自然膜状の環境から単離されたタンパク質である。したがって、例えば、ウイルス糖タンパク質の高次アーキテクチャなどの重要な詳細は、ビリオンの文脈において、欠けているままである。一方、電子低温顕微鏡法および単一粒子再建全体のエンベロープウイルスの正20面体対称17,18とビリオンに制限されています。サブボリュームの配置と組み合わせた電子cryotomographyは、このように、その場で不規則な形状、多形性ウイルスの糖タンパク質スパイクの研究を可能にする補完的な技術として浮上している。 私たちは、Jsubtomo断層サブボリュームの検出、位置合わせ、および平均化（www.opic.ox.ac.uk/jsubtomo）というソフトウェアを開発しました。 Jsubtomoは細胞およびウイルスの構造19-26数の構造決定に利用されている。ここでは、決定、ウイルス表面のスパイク構造を可能にする詳細なプロトコルを概説しています。ノイズを相関させることによって、過剰洗練平均した構造の回避するために、「ゴールドスタンダード」の改良方式が10,27を採用しています。最後に、典型的な結果の可視化と解釈のための戦略が議論されている。

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jsubtomo (ver 1.3.1)</td>
			<td style="width:184px;">University of Oxford</td>
			<td style="width:119px;">n/a</td>
			<td>www.opic.ac.uk/jsubtomo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bsoft (ver 1.8.7)</td>
			<td style="width:184px;">NIAMS, NIH</td>
			<td style="width:119px;">n/a</td>
			<td>bsoft.ws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UCSF Chimera</td>
			<td style="width:184px;">UCSF</td>
			<td style="width:119px;">n/a</td>
			<td>www.cgl.ucsf.edu/chimera</td>
		</tr>
	</tbody>
</table>

averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using jsubtomo

エンベロープウイルスは、宿主細胞への侵入を媒介するそれらの表面上に膜糖タンパク質を利用する。これらの糖タンパク質「スパイク」の三次元構造解析は、多くの場合、ウイルスの病原性を理解するための薬物設計における技術的に困難だが重要である。ここで、プロトコルは、電子クライオ断層撮影データの計算、平均化を介してウイルスのスパイク構造決定のために提示されている。クライオ電子断層撮影は、ネイティブに近い、凍結水和状態における膜ウイルスなど多形の生物学的標本の3次元断層ボリューム再構築、または断層像を導出するために使用される電子顕微鏡の技術である。これらの断層像は、低解像度ではあるが、三次元の関心の構造を明らかにする。サブボリューム、またはサブ断層像の計算平均、例えば、ウイルス糖タンパク質スパイクなどの構造モチーフを、繰り返しの高解像度の詳細を取得する必要がある。アリのための詳細な計算アプローチgningとJsubtomoソフトウェアパッケージを使用してサブ断層像を平均化することが概説されている。このアプローチは、ビリオン膜上の高次スパイク·ツー·スパイクの相互作用の研究の20〜40オングストロームの範囲の解像度と研究にウイルス糖タンパク質スパイクの構造の可視化を可能にします。典型的な結果をBunyamweraウイルス、 ブニヤウイルス科からのエンベロープウイルスのために提示されています。このファミリーは、ヒトおよび動物の健康に脅威を与え、病原体の構造的に多様なグループです。

私たちは、24セット 、以前に公開されたデータを使用してBunyamweraウイルス（Orthobunyavirus、 ブニヤウイルス科 ）のエンベロープ糖タンパク質複合体について上記に概説されたサブ断層アベレージングワークフローの適用を実証する。データ収集および精密化パラメーターを表1に示す。一つの代表的断層像は、 図2に示されている。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> パラメータ（単位） </td><td> 値 </td></tr><tr><td> データ収集 </td><td></td></tr><tr><td>電圧（kVの） </td><td> 300 </td></tr><tr><td>キャリブレーション済みの倍率（X） </td><td> 111000 </td></tr><tr><td>画素サイズ（Å） </td><td> 5.4 </td></tr><tr><td>推定線量（E - /Å2） </td><td> 100 </td></tr><tr><td> Underfocus（μm）と</td><td> 4.0〜4.5 </tD&gt; </tr><tr><td>まずCTFゼロ（Å）A </td><td> 26-30 </td></tr><tr><td>チルト範囲（°） </td><td> -60-60 </td></tr><tr><td>チルトサンプリング（°） </td><td> 3 </td></tr><tr><td> データと洗練 </td><td></td></tr><tr><td>断層像</td><td> 11 </td></tr><tr><td>ビリオン</td><td> 29 </td></tr><tr><td>ビリオンあたり種子</td><td> 106 </td></tr><tr><td>種子の総数</td><td> 3074 </td></tr><tr><td>検出されたスパイク</td><td> 1,346 </td></tr><tr><td>重複を除去した後スパイク</td><td> 1,401 </td></tr><tr><td>相互相関に基づく選択後にスパイク</td><td> 1022 </td></tr><tr><td>最終的な平均に含まスパイク</td><td> 1022 </td></tr><tr><td>対称性</td><td> C3 </td></tr><tr><td>角度サンプリング（°） </td><td> 8 </td></tr><tr><td>解像度洗練で使用範囲（Å） </td><td> 42から334 </td></tr><tr><td>最終的な解像度の見積もり（Å）B </td><td> 35 </td></tr></tbody></table> 表1：Bunyamweraデータ収集及び精密化統計。 CTF、コントラスト伝達関数。 B 0.143の閾値で2つの独立して洗練された構造の間のフーリエシェル相関を用いて算出。 <img alt="図2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> 図2：Bunyamweraビリオンの断層を通じてスライス各ビリオンの周辺で明らかにいくつかのスパイクサイドビューは矢印で示されている。断層像は、低域60オングストローム濾過された。スケールバーは100nm。 まず、205手動で選んだスパイクを使用して初期モデルを（精製<strong&gt;図3）。最も中心にあるスパイクの三回対称性は、任意の対称性（ 図3B）を適用せずに明らかであったと洗練のその後のラウンド（ 図3C）に課された。ビリオン表面上に自動的にすべてのスパイクを検出するために、私たちは43 nmおよび20度（ 図4A）の間隔の半径でそれぞれビリオンのための106の種を生成し、反復して膜（ 図4B）の相対的な位置を洗練。 <img alt="図3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> 図3：初期テンプレート構造の改良。 （A）は円筒はスパイクの手動で定義された位置から構築（C100）のテンプレートを平均した。（B）いずれかの対称性（C1）のない洗練された5回の三倍を付けて表示しスパイクを課した後の濃度を平均化対称特徴。モデルの解像度は48オングストロームである。スパイクの（C）の平均は、3回対称性を持つ洗練された5ラウンド後に41Åで決議した。 <img alt="図4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> 図4：種子の改良 （A）と（b）の後に洗練前の種子のAB）のサブセットは、図2から1ビリオン密度に示されている（B）中の種子は、それぞれのクロスに基づいて色分けされています相関係数（青、低い相関;赤、高い相関）。 （重複を除去した後、トップ75％;〜千スパイク）最高の相関糖タンパク質スパイクパッチは、最終的な平均を計算するために使用した。平均値は、35オングストロームの分解能（ 図5）に解決されました。それは、途中で三量体スパイク構造を明らかにした6近隣のスパイクからいくつかの貢献に加えて。既知の位置に構造を配置することによって計算されたビリオンの複合モデルは、（ 図6A）、ビリオン表面上のスパイクの配置を明らかにした。時折、スパイクのローカルに順序付けられたパッチは、（ 図6B）明らかであった。 <img alt="図5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> 図5：テンプレートマッチング後の糖タンパク質スパイク層のパッチの洗練された構造。 「偶数」および「奇数」（AB）2マップデータの両半分からの再構成が示されている。 2マップは、アプローチの妥当性を検証する、注目すべき程度の類似性を示している。 2マップは、互いに完全に独立して再構築さ​​れてきたように、スパイク長軸周りの向きが異なっている。 （C）は、2つのマップの平均はで示されている35オングストロームの最終的な解決。 <img alt="図6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> 図6：Bunyamweraビリオン上のスパイクの配置。 （A）ビリオンの複合モデル。ビューベクトル（スティック）がスパイクの向きを示している。色は各スパイクやテンプレート構造間の相互相関を示している。（青、低い相関;赤、高い相関を）、（B）のスパイクの順序付きパッチのクローズアップ。

Watch this Scientific Journal Video about Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo at JoVE.com

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomoを用いた電子Cryotomography再建からのウイルスのエンベロープ糖タンパク質スパイクの平均化

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

averaging-viral-envelope-glycoprotein-spikes-from-electron

Research

JoVE Journal

Immunology and Infection

12.0K ビュー.  University of Oxford. An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

ビデオ: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

ウイルス性病原体のために臨床サンプルをスクリーニングするためにパンウイルスマイクロアレイアッセイ（Virochip）を使用して、

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

免疫・感染学

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

低温電子断層撮影と自動化されたサブ断層アベレージングを使用して、HIVエンベロープ糖タンパク質の分子構造の決定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

特定のヒトと動物のウイルスを用いた微生物ソースのトラッキングのための費用対効果の高い方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

ウイルスの変異頻度のRNAウイルスと特性の忠実性の変異の分離

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

ハイドロキシアパタイトクロマトグラフィーを用いた環境ウイルスのコミュニティから一本鎖DNA、二本鎖DNAとRNAの分離

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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. ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

合成脂質二重膜上でHIV-1エンベロープ誘発性ウイルス学的シナプスとシグナリングのイメージング

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

細胞ベースのELISAアッセイを使用して、HIV-1エンベロープ糖タンパク質三量体の構造評価

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

の検出のための凍結乾燥LAMP法試薬の開発<em&gt; Q熱リケッチア</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

酵素結合レクチンアッセイによってインフルエンザノイラミニダーゼ阻害抗体価を測定します

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...