作業は、DP2 OD004631とR01 AI084119を含む、ビルアンドメリンダゲイツ財団（＃38631）とNIHからの補助金によってサポートされていました。

1。方法<ol><li>プロトコルの最初のステップでは、in silicoでフォールドするV3王冠のシーケンスを選択することです。 R2の株について、このフラグメントのシーケンスはKSIPMGPGRAFYTです。 </li><li>この配列に相当するペプチドの三次元原子構造は、コンピュータの仮想空間に構築する必要があります。このためにICMのコマンドは次のとおりです。 buildpep&quot;KSIPMGPGRAFYT&quot; または[ファイル：新規：ペプチドは、プルダウンメニューで選択することができます</li><li>手順のいくつかのパラメータは、検索のステップ数（検索の長さ）、シミュレーションの温度、バイアス合理的な分野、エネルギーの観点から、異なるエネルギーの計算方法の選択に向かって検索を変数に制約を含む検索戦略パラメータを含む、設定されていますと検索がこのようなムービーを記録するかどうかとして記録し、どのように多くの中間得点コンフォメーションする方法を示すパラメータが記録されることが望ましい。すべてのこれらのパラメータは、以前の出版物（www.molsoft.comを参照）によって最適化されています。 </li><li>折りたたみは、次のコマンドを使用して開始されます。 モンテカルロ または[ファイル：新規：ペプチドは、メニュー分子力学プルダウンで選択することができます。最小化：グローバル 前者の場合には、ステップ3からのパラメータは、コマンドラインで一つ一つ設定する必要があります。コマンドが実行される前に、後者のケースでは、最も一般的に選択したパラメータのチェックボックスがペインに提供されています。便宜上、この実験と以前に公表された実験に利用したのと同じ折りたたみICMのスクリプトはここに示されています： <img src="/files/ftp_upload/2118/2118output.jpg" alt="出力" /> このスクリプトは、テキストフ​​ァイルに保存され、急速にコマンドを使用してコンピュータのオペレーティングシステムのコマンドライン（通常、Linux）から実行できます。 ICM _foldingscript </li></ol> 2。成功の秘訣<ol><li> V3ループは、ほとんどすべての既知の菌株の長さが一定の35アミノ酸であり、アミノ酸の位置は1でシスチン結合元のジスルフィドから始まり、35でシスチン結合に対応するジスルフィドで終了する1〜35に番号が付けられていますので。それがループの一部なので、V3の王冠の境界は慎重に選択する必要があります。大きすぎるフラグメントが選ばれているなら、それは遊離ペプチドであるかのように自由に柔軟なセグメントとして動作する可能性は低い、折り畳みシミュレーションが正しく構造を評価しないように。小さすぎるフラグメントが選択されている場合は、有益な三次構造は、シミュレーションで形成されないことがあります。私たちの以前の研究では、位置10から位置22へのフラグメントは、抗体結合結晶コンフォメーションと相関することを示したので、これは折り畳みのために選択する必要のあるすべてのV3ループの断片です。 </li><li>グラフィカルユーザーインタフェースを使用しているがとプルダウンメニューは、ICMとスクリプト上で使用した特定のV3ループの王冠のフォールディングを使用して、一般的にペプチドの折りたたみに関するユーザーフレンドリーな、成功する前の仕事、単にbuildpepラインにシーケンスを変更および実行することです。コマンドラインから上記スクリプトの使用をお勧めします。 </li></ol> 3。代表的な結果 R2の折りたたみの結果は任意のV3ループの結果の代表的なものである。結果を評価するために、プロジェクトファイルは、（それが上記のスクリプトから&quot;newProject1.icb&quot;という名前になります）選ばれた&quot;と分子力学、スタックは、表示&quot;を開き、必要があります。スタックのコンフォメーションの表が表示されます。スタックのコンフォメーションは、プロット/ヒストグラムアイコンをクリックして、グラフィカルに可視化することができる。 &quot;分子力学、スタック、プレイは&quot;視覚的に折りたたみ式によって発見された立体配座の好みを理解するためにスタックのムービーを作成します。 α-ヘリックスのコンフォメーション、特に明確なβ-ストランドの設定がスタック全体に見られる12から14の位置にある断片で、そして非常に少数- R2のシーケンスの場合は、コンフォメーションは、β-ヘアピン等のV3ループ2に期待されている見られている。さらに、約3単位のエネルギーギャップは、最低エネルギーコンフォメーションと番目に低いエネルギー配座の間に見られる。エネルギーの観点から、この手段時間の1％未満の最低エネルギー配座の構造のみがちらつくていること：折り畳み式の結果は、したがって、R2 V3王冠が硬いのではなく、柔軟性、構造であることを示唆している。そこにコンフォメーションのアンサンブルにおける他の重要な構造的特徴であるが、それらは体系的に理解することが困難である場合があります。 <table border="1"><tbody><tr><td></td><td> JRFL </td><td> SF162_V3JRFL </td><td> SF162_V3R2 </td></tr><tr><td> 447 - 52D GMT 50 </td><td> 15 </td><td> 0.00061 </td><td> 0.00078 </td></tr></tbody></table> 表1：マスクとマスクされていない設定でJRFLとR2 V3ループの相対的な中和。データは、以前はカルドソT.、 ら。ARHR（200で報告された8）とピンター、A.、 ら。ら J. Virol（2004）。 SF162_V3JRFLが含まれていますJRFL：簡単に言えば、中和活性は、env -欠陥ルシフェラーゼ発現JRFL Envのまたは上記SF162 V3の亜種でシュードpNL4 - 3.Luc.RE -プラスミドで生成されたpsVsを使用して単一サイクルの感染性試験により測定したSF162 V3のループシーケンスの代わりにV3のループシーケンス。 SF162_V3R2はSF162 V3のループシーケンスの代わりにR2のV3のループシーケンスが含まれています。 psVsは、37で1.5時間のための447 - 52D mAbの連続希釈液° Cをインキュベートし、ポリブレンの存在（10 mgの= mL）を96ウェルプレートに播種CD4 + CCR5 + U87標的細胞に添加した。 24時間後、細胞をインキュベーションの追加の24〜48時間で10％FBSおよび10 mg = mLのポリブレンを含むRPMI培地でrefeedの過去過去分詞形れました。ルシフェラーゼ活性はプロメガからアッセイ試薬を用いてマイクロタイタープレートルミノメーター（HARTA、（株））と48〜72時間の感染後に決定された、447 - 52D 50％の中和（GMT50）のための（株）幾何平均力価は、中和曲線から補間により決定され、少なくとも3つの独立したアッセイの平均値である。

<ol>
	<li>Almond, D., Kimura, T., Kong, X., Zolla-Pazner, S. &. a. m. p. ;. a. m. p., Cardozo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+characterization+of+the+V3+loop+crown.">Dynamic characterization of the V3 loop crown.</a> Antiviral Therapy. 12, 13-31 (2007).</li><li>Almond, D., Kimura, T., Kong, X., Swetnam, J., Zolla-Pazner, S., Cardozo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+conservation+predominates+over+sequence+variability+in+the+crown+of+HIV+type+1's+V3+loop.">Structural conservation predominates over sequence variability in the crown of HIV type 1's V3 loop.</a> AIDS Res Hum Retroviruses. 26, (2010).</li><li>Abagyan, R., Totrov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+probability+Monte+Carlo+conformational+searches+and+electrostatic+calculations+for+peptides+and+proteins.">Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins.</a> J Mol Biol. 235, 983-1002 (1994).</li><li>Quinnan, G. V., Zhang, P. F., Fu, D. W., Dong, M., Alter, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+HIV+type+1+envelope+protein+associated+with+a+broadly+reactive+neutralizing+antibody+response.">Expression and characterization of HIV type 1 envelope protein associated with a broadly reactive neutralizing antibody response.</a> AIDS Res Hum Retroviruses. 15, 561-5670 (1999).</li><li>Zhang, P. F., Bouma, P., Park, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variable+region+3+(V3)+mutation+determines+a+global+neutralization+phenotype+and+CD4-independent+infectivity+of+a+human+immunodeficiency+virus+type+1+envelope+associated+with+a+broadly+cross-reactive,+primary+virus-neutralizing+antibody+response.">A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4-independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response.</a> J Virol. 76, 644-655 (2002).</li></ol>

利害の衝突は宣言されません。

のab initioの折りたたみは、三次構造上の特性、順次個々のアミノ酸の研究から明らかでないかもしれないの効果を明らかにする。 R2 V3ループ王冠のいくつかの以前に無名の構造特性は、フォールディングシミュレーションによって明らかにされています。これらの観測された構造的な好みは、中和とhyperinfectivity 5〜感度すなわち、R2のひずみの観測機能的特性と相関すること?...

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<td>ICM-Pro</td>
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assessment of immunologically relevant dynamic tertiary structural features of the hiv-1 v3 loop crown r2 sequence by ab initio folding

HIV - 1の抗原の多様性は、長い間ワクチンの設計への障害となっており、この変動は、特にウイルスの表面のエンベロープ糖タンパク質のV3ループの中で発音されます。我々は以前、ダイナミック、シーケンス変数が、V3ループの王冠と提案した免疫学的に関連するβ-ヘアピン三次構造へのHIV - 1ウイルスの人口全体に制約されます。重要なのは、別のV3ループの王冠のシーケンスの数千は結晶学やNMRによることが困難または不可能なウイルスの多様性全体の動向の3次元構造解析を行う、HIV - 1ウイルスの循環でもあります。 V3王冠1、2の折り畳みと私たちの以前の成功した研究では、ICM - Proの分子モデリングのソフトウェアパッケージ（Molsoft LLC、ラホーヤ、カリフォルニア州）でアクセス可能なのab initioのアルゴリズム3を使用して示唆されたV3ループの王冠、特にから位置10から22、それは溶液中で自由に折りたたみ式の制約のないペプチドであるかのように大規模な程度に動作するように十分にその隣接茎の柔軟性と長さから利益を得る。 60,000個々の株のV3ループのちょうどこの部分のような、急激なのab initioフォールディングとして+循環HIV - 1株は、有益なことができます。ここで、我々は、そのユニークな特性の構造的基盤への洞察を得るためにR2株のV3ループを折ら。 R2は、患者の血清およびモノクローナル抗体4,5による中和にこの株の絶妙な感度の責任であると考えられる稀なV3のループシーケンスを負いません。ひずみCD4非依存性感染症を媒介とは、広く中和抗体を誘発するために表示されます。我々は、折りたたみの結果の評価がR2のために観察された免疫学的活動と折りたたみで観測された構造を関連付けるための有益なことができる方法を示しています。

Watch this Scientific Journal Video about Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding at JoVE.com

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

HIV - 1の表面のエンベロープ糖タンパク質（gp120の）の別のV3のループシーケンスの冠の領域は、構造的にポジションのインシリコのフォールディングにによって多くのケースで特徴づけることができる最新鋭のを使用してループの10から22<em&gt;第一原理</em&gt;折りたたみア​​ルゴリズム。ここでは、HIV - 1のR2株、不可解な機能特性を持つユニークな中和感受性株からのV3ループのこの地域の折りたたみと評価を示しています。

によるHIV - 1 V3ループクラウンR2シーケンスの免疫学的に関連する動的第三紀の構造的特徴の評価第一原理折りたたみ

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

assessment-immunologically-relevant-dynamic-tertiary-structural

Research

JoVE Journal

Immunology and Infection

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

9.5K ビュー.  School of Medicine, New York University. HIV - 1の表面のエンベロープ糖タンパク質（gp120の）の別のV3のループシーケンスの冠の領域は、構造的にポジションのインシリコのフォールディングにによって多くのケースで特徴づけることができる最新鋭のを使用してループの10から22第一原理折りたたみア​​ルゴリズム。ここでは、HIV - 1のR2株、不可解な機能特性を持つユニークな中和?...

ビデオ: Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

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 ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

標準的な臨床アッセイによる検出限界以下のウイルス量とHIV感染個体におけるHIV - 1 RNAを増幅して定量化

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

クライオ電子顕微鏡によるHIV - 1キャプシドアセンブリの構造とヘリカル実空間の反復再構成

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

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 ...

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 ...

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 ...

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 ...

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. ...