私たちは彼の寛大なA32の贈り物、17bと、48D、およびC11 mAbの博士ジェームズ·ロビンソンに感謝します。 PGT 121は、NIH AIDS試薬プログラム、エイズ、NIAID、NIH（カタログ番号12343）の事業を通じて得られた。この作品は、科学者が、AFにしてCRCHUM連続グラントによってだけでなく、CIHR触媒グラント＃126630によって＃24639を付与ヤングのFRQS設立により、CIHRオペレーティング＃257792により、イノベーション拠点リーダーの＃29866のためのカナダ財団によってサポートされていましたAFとMRへ。 AFはFRSQ Chercheur Boursierジュニア1フェローシップ＃の24639の受信者である。 MVはCIHR博士研究賞の＃291485によってサポートされていました。

1 1日目 - 細胞培養<ol><li>プレート2×10 4ヒト骨肉腫（HOS）発光読み取りに適した不透明な96ウェル細胞培養プレート中のウェルあたりの細胞。 10％ウシ胎児血清（FBS）および100 U / mlペニシリン - ストレプトマイシンを補充したダルベッコ改変イーグル培地（DMEM）を使用する。 37°C、5％CO 2で翌日までインキュベートする。 </li></ol> 2 2日目 - ポリエチレンイミン（PEI）トランスフェクション<ol><li>以降の手順に従って、トランスフェクションミックスを調製する。同じのEnvでトランスフェクトされるべきである井戸の数に応じて試薬とDNAの量を調整します。 </li><li>チューブA：25mMのHEPESを補充したDMEMμlの5に10ngのTatのコード（例えばPTAT-III 27など）は、プラスミドと150ngのEnvのコードプラスミドを追加します。 </li><li>などpSVIIIなどのTat依存のEnvをコードするプラスミドを使用した場合のTat-コードプラスミドにのみ必要です。 </li><li>チューブB：1から（450 ngのPEIを追加5μlのDMEM中にμgの/μlの溶液）。 </li><li>徹底的に10秒間ボルテックスチューブA.ミックスにチューブBの内容を追加し、トランスフェクション混合物を室温（22℃）で10分間インキュベートする。 </li><li> 96ウェルプレートのウェルあたりトランスフェクション混合物10μlを追加します。 37°C、5％CO 2で48時間インキュベートする。 </li></ol> 3。4日目 - ELISA <ol><li>のEnv /抗体複合体の可能性エンドサイトーシスを最小限にするために、室温ですべての実験を行います。 </li><li>同時に使用されているプレートあたりの洗浄バッファー250ミリリットルを用意してください。洗浄緩衝液は、1×トリス緩衝食塩水（TBS）pH7.5である（50 mMトリス-Clを、pH7.5の、150mMのNaCl）、1mMのMgCl 2および1.8mMのCaCl 2を補充した。 </li><li> 1％脱脂粉乳および洗浄緩衝液に5 mMトリスpH8.0のを追加することによって、プレートあたりブロッキングバッファー125ミリリットルを用意します。 </li><li> 96ウェルプレートから細胞培養培地およびトランスフェクション混合物（上清）を除去する。 </li><li> 100μの追加;ウェルあたりブロッキングバッファーし、室温で20分間インキュベートリットル。 </li><li>上清を除去し、ブロッキング緩衝液で適当な濃度に希釈ウェル当たり抗体（または血清）を50μlを追加。一般的に、11μg/ mlの濃度を使用しています。室温で1時間インキュベートする。 </li><li> 100μlのブロッキング緩衝液、その後、100μlの洗浄緩衝液で洗浄工程を3回繰り返すとウォッシュ3倍。 </li><li>上清を除去し、ブロッキング緩衝液を100μlを追加し、室温で5分間インキュベートする。 </li><li>上清を除去し、ブロッキング緩衝液中で1/3000に希釈した二次抗体を50μlを追加。メーカーの違いに応じた最適な抗体希釈を変更します。 RTで40分インキュベートします。 </li><li> 100μlのブロッキング緩衝液、その後、100μlの洗浄緩衝液で洗浄工程を3回繰り返すとウォッシュ3倍。 </li></ol> 4。データ収集<ol><li>プレートから上清を除去し、ウェル当たり30μlの1×高感度化学発光（ECL）基板を追加します。 </li><li> CHEMを取得1秒間iluminescence信号/ウェル上の製造者の指示に従って、適切なプレートリーダー。時間を読むことは、ハードウェアの違いにより異なる場合があります。 </li></ol>

<ol>
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著者らは、利害の衝突を宣言しません。

このアッセイは、HIV-1 Envの三量体が細胞表面で発現を特異的mAbの相互作用を検出するために最適化される。プロトコルが確立されると、それは全体的に低い材料コストおよび抗体の少ない量で高いスループットを培地で使用することができる。このアッセイは、トランスフェクションに基づいているので、容易にEnvのコンホメーションに及ぼす影響を研究するために、CD4のような細胞タンパ?...

三量体ウイルスエンベロープ糖タンパク質（Envの）によって媒介されるヒト免疫不全ウイルス1型（HIV-1）のエントリは、感染サイクルの第一段階である。ビリオンの表面に提示のみさらさウイルス抗原なので、Envの三量体は、中和および非中和抗体を誘発する。このように、ワクチンの免疫原の設計のための興味深い候補を表わす。しかし、可溶性または組換え形態におけるのEnvを用いたワクチン接種試験は最も主要なHIV-1に対する最小限の有効性応答を誘発し1-3を分離します。それにもかかわらず、RV144ワクチン臨床試験4で観察された部分的な有効性は、免疫原候補として、HIV-1のEnvに興味をリニューアルしました。これは、ワクチン誘発抗-ENV抗体はSIVとHIV 5挑戦に対する保護のある程度を生成するのに十分であったことを記述した最近の研究によって確証された。 小胞体内で合成された後、Envのglycoprote前駆体のgp160に、ウィルス融合プロセスを燃料する能力にとって重要なさまざまな翻訳後修飾を受ける。のEnv前駆体は、gp120-gp41の ​​リエゾンを維持し非共有相互作用で、その細胞質外gp120およびgp41の ​​膜貫通サブユニット6-10に切断される前に、三量体で適切に関連付ける倍なければなりません。感染した細胞の機構も大きく、その総質量11,12の約​​50％を占めたEnvをグリコシル化する責任があります。良い異なるコンホメーションを理解することの重要性を強調し、Envのが適応し、それ以外の場合は15〜19を暴露される特定の高度に免疫原性エピトープを非表示にすることを可能にすると考えられている準安定性を提供しながら、得られた複合体構造は、Envのは13,14立体構造的に柔軟であることを可能にするネイティブのEnv三量体によってサンプリング。 今日までに、いくつかの技術が開発され、首尾よくEnvのコンホメーションchのを研究するために使用されているアンジェス。しかし、それらは多くの場合、特定のEnvコンテキストに限定されるものでは、その限界が異なります。例えば、表面プラズモン共鳴またはコンフォメーション特異的モノクローナル抗体を用いた免疫沈降アッセイ（mAb）は、どちらかは、それらの三量体形態20,21からの免疫遺伝学的に異なることが知られている単量体の可溶性または可溶化のEnv分子に依存している。最近の研究ではまた、切断は主に抗体14,22,23を非中和により認識されるエピトープの露出をもたらしたEnvのコンフォメーションに影響を与えることを示唆している。 ここでは詳細にcellularly発現さのEnv三量体18,24-26のコンフォメーションの迅速かつ容易な決定を可能にする方法が記載されている。ヒトの付着性細胞株におけるのEnvの一過性トランスフェクション後のEnv特異的抗体の結合は、単純な化学発光反応を用いて検出される。この技術は、コンフォメーションdepenの立体配座優先性を特徴付けるために使用することができる凹み抗体。したがって、このアッセイは、堅牢で柔軟性の高い検出方法を提供する。

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			<td height="21" style="height:21px;width:216px;">HOS cells</td>
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			<td height="42" style="height:42px;width:216px;">White Opaque Tissue Culture Plate, 96 well, Flat Bottom</td>
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			<td height="42" style="height:42px;width:216px;">Polyethylenimine, linear, 25000 MW</td>
			<td style="width:103px;">Polysciences</td>
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			<td>Prepared in 1mg/ml solution</td>
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			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:103px;">Invitrogen</td>
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			<td height="42" style="height:42px;width:216px;">Goat Anti-Human IgG, Peroxidase Conjugated</td>
			<td style="width:103px;">Pierce</td>
			<td align="right" style="width:119px;">31413</td>
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			<td style="width:103px;">PerkinElmer</td>
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			<td style="width:103px;">Berthold Technologies</td>
			<td style="width:119px;"></td>
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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.

上記した一般的手順を用いて、野生型（wt）又は変異EnvのいずれかでたCD4iエピトープの露出に対する可溶性CD4（sCD4を）と共発現細胞のCD4の影響をアッセイするためのプロトコルを適合18,24前述したように、 25,28。 図1は、一般的な手順とsCD4を持つまたは携帯CD4 18の共発現による治療後のたCD4iエピトープの露出を表しています。 図2では、私た...

Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

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

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 ...

conformational-evaluation-hiv-1-trimeric-envelope-glycoproteins-using

Research

JoVE Journal

Immunology and Infection

14.3K ビュー.  Université de Montréal. 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.

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

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

緑色蛍光タンパク質を発現するリビジョンに依存するレンチウイルスベクターを用いたHIV - 1陽性細胞の特定のマーキング

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

免疫・感染学

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3の人口ベースのシーケンシングを用いたHIV - 1トロピズムの遺伝子型推論

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

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 ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

遺伝子型アプローチを用いた配列解析によるHIV-1コレセプター使用（指向性）の予測

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

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 ...

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

ヌクレオカプシドアニーリング媒介電気泳動（NAME）アッセイは、HIV-1ヌクレオカプシド阻害剤の迅速な同定を可能にする

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

ハイスループット定量的リアルタイムRT-PCRアッセイのタイプIおよびIIIインターフェロンサブタイプの発現プロファイルを決定するための

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

HIV-1陽性個体の血漿からエキソソームの単離

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...