作業は、NIAIDからYWの公衆衛生サービス助成金1R01AI081568でとM.ローゼンと2日目（株）主催の2009 NYCDCエイズライドのドナーとライダーの寛大な寄付によって部分的にサポートされていました

1。 REV -依存性のウイルス粒子の生産のためのプラスミドのトランスフェクションセットアップ：REV -依存GFPレンチウイルスベクター、PNL - GFP - RRE - SAは、以前に4,5,6に記載した。ウイルス粒子にアセンブルするために、プラスミドは、HIV - 1のパッケージ構造、pCMVΔ8.2（親切に先生Dider Tronoで提供される）、およびVSV - Gの糖タンパク質を（pHCMV - G）を有するプラスミドとHEK293 T細胞にcontransfectedした。トランスフェクションは、リン酸カルシウム法を用いて行った。 バッファーの調製：10 × HBSは、（HEPES緩衝生理食塩水）Hepes緩衝5gを溶解して調製した、8塩化ナトリウムのg、100 mLの中のNa 2 HPO 4（無水）のKCl、ブドウ糖1gの、0.103グラムの0.37グラムH 2 O注しthe10 X HBSの1mLのアリコートにバッファと-20℃で保存トランスフェクションの時に、H 2 O（1:5希釈）で2 X HBSに10 × HBSを変換する、と7.05の間にpHを調整する- 7.12。 （10mLの2 × HBSのために、それは通常1MのNaOHの約50μLが必要です）。 0.22μmのミリポアフィルターを通過することで2X HBSバッファーを滅菌フィルタします。 CaCl 2のバッファは、最終濃度が2Mに10mMのHEPESでCaCl 2を溶解させることにより行われた。 5.8にpHを調整し、4でバッファとストアを滅菌フィルタ℃を手順： <ol><li> 100ミリメートルペトリ皿、および37℃で10mLのDMEM + 10％FBSで一晩細胞を成長° C、5％CO 2のトランスフェクションの1日前、シード2 × 10 6 HEK293T細胞。 </li><li>翌日、細胞からの上清を除去し、5 mLの新鮮なDMEM + 10％FBS、4時間連続的に培養細胞で置き換えてください。 </li><li> 5mLのポリスチレンチューブに480μL2X HBSバッファーを添加します。 </li><li>目の5 mLのポリスチレンチューブに480μLの合計量を60μL2M CaCl 2のバッファ、プラスミドDNA、およびトランスフェクションTEバッファー（1mMトリス、25mMのEDTA、pH8.0）を加える。各ペトリ皿の場合、プラスミドはPNL - GFP - RRE - SA、pCMVΔR8.37.5μgの、とpHCMV - Gの2.5 mgの10μgの割合で添加する必要があります。 </li><li> 2 X HBSのチューブにプラスミドDNA - CaCl 2の混合物の滴下を追加し、室温で30分間インキュベートする。微細な析出物が形成されます。 </li><li>細胞にピペットで滴下して混合物の960μLを加える。 37℃、一晩5％CO 2を 。 </li><li>翌日、上清を除去し、10mLの新鮮なDMEM + 10％FBSを追加し、連続して37℃、5％CO 2で一晩細胞をインキュベートする。 </li><li>ポストトランスフェクション48時間後に、上清を除去し、50mLの滅菌チューブにそれを転送することにより、ウイルスを収穫。各ペトリ皿に新鮮な10mLの新鮮なDMEM + 10％FBSを追加し、一晩培養し続ける。 4でウイルス上清℃にて保存してください。 </li><li>上清を収集することで、72時間後にトランスフェクションで収穫を続ける。すべての上清を組み合わせ、そしてペレットに15分間500 × gで上清を遠心し、細胞の破片を取り除く。 </li><li>上清を0.22μmのミリポアフィルターを介して収集し、ろ過した。ウイルスは、さらにサイズ排除カラムを通して濃縮した。 </li></ol> 2。ウイルス粒子を集中し、ウイルス力価を決定する<ol><li>ウイルス粒子は、サイズ排除カラム（100,000 MWカットオフ、Centricoin）により濃縮した。ウイルス上清をカラムにロードされ、4 15分間℃で6000 × gで遠心分離した。 </li><li>濃縮されたウイルス上清を回収し、分注し、-80℃で保存した。 </li><li>ウイルス力価は、TNF -治療、HIV - 1陽性ジャケットの細胞株、J1.1 7の感染によって決定した。細胞を、1ml当たり2.5 × 10 5細胞（100mLの総容積）を96ウェルプレートで培養し、週のための希釈VNL - GFP - RRE - SAの100mLで感染させた。粒子の力価（TCID50）はリードおよびMuench 8の方法に従ってGFP陽性ウェルを計数することにより推定された。 </li></ol> 3。レンチウイルスパーティクルとマーキングHIV - 1陽性細胞、VNL - GFP - RRE - SA セットアップ：ヒトCD4 T細胞株、CEM - SSは、最初のHIV - 1を感染させた。 48時間後の感染では、細胞がレンチウイルス粒子、VN​​L - GFP - RRE - SAとの重複感染した。さらに2日間の重複感染に続いて、GFP陽性細胞はフローサイトメトリーによって分析した。対照として、HIV - 1感染CEM - SS細胞は同じVNL - GFP - RRE - SAに感染させた。唯一のHIV - 1感染CEM - SS細胞はGFP陽性細胞ではなく、HIV - 1感染CEM - SS細胞をもたらした。 手順： <ol><li>感染2時間前には、最終濃度4 mg / mlにpolybraneを追加。細胞をカウントし、各感染症のために2 × 10 5細胞を取る。 2時間HIV - 1 NL4 - 3の200 ngの（P24）で細胞を感染させる。 </li><li> 10分を300 × gで遠心分離により細胞を洗浄、再懸37 2 × 10 5細胞/ mL、そして文化℃、48時間に細胞。 </li><li>細胞をカウントし、VNL - GFP - RRE - SAでの感染あたり2 × 10 5細胞を取る。 37℃で一晩VNL - GFP - RRE - SAとインキュベート100 mLを加え。細胞を洗浄し、2 × 10 5細胞/ mLに懸濁します。 </li><li> 48時間後の重感染でフローサイトメトリー分析を行います。感染細胞をペレット化し、20分間室温でインキュベート500mLの1％paraformaldehede、中に再懸濁し、そしてその後FACスキャンアナライザー（ベクトンディッキンソン）で分析した。 </li></ol> 4。代表的な結果実験が正常に実行されている場合、コントロールに、GFP陽性細胞がHIV - 1感染CEM - SSで検出されず、一方で、かなりのGFPの人口は、フローサイトメーターによるHIV - 1感染CEM - SS細胞で検出されます。細胞。 実験が正常に実行されている場合、REV -依存レンチウイルスベクター、VNL - GFP - RRE - SAは、緑色蛍光タンパク質（GFP）の発現の測定により、細胞集団を生体内で複製HIVの非常に特異的な検出が可能になります。この例では、収穫されたCEM - SS細胞は、マウスCD24、HSAに対するPE標識ラットモノクローナル抗体で染色し、その後、HSAとGFP発現の両方のフローサイトメーターで分析。 <img src="/files/ftp_upload/2198/2198fig1.jpg" alt="図1" /> 図1ここで示したように、かなりのGFPの人口がVNL - GFP - RRE - SA（図1c）との重複感染HIV - 1感染細胞で検出された。対照的に、GFP陽性細胞はいずれのHIV - 1感染CEM - SS細胞にレンチウイルスの重複感染（図1a）またはレンチウイルスの重複感染によるHIV - 1感染細胞（図1b）なしでは検出されなかった。

<ol>
	<li>Garcia, J. A., Wu, F. K., Mitsuyasu, R., Gaynor, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+cellular+proteins+involved+in+the+transcriptional+regulation+of+the+human+immunodeficiency+virus.">Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.</a> EMBO Journal. 6, 3761-3770 (1987).</li><li>Merzouki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+gp120/160+expressing+cells+upregulate+HIV-1+LTR+directed+gene+expression+in+a+cell+line+transfected+with+HIV-1+LTR-reporter+gene+constructs.">HIV-1 gp120/160 expressing cells upregulate HIV-1 LTR directed gene expression in a cell line transfected with HIV-1 LTR-reporter gene constructs.</a> Cell Mol Biol (Noisy-le-grand. 41, 445-452 (1995).</li><li>Aguilar-Cordova, E., Chinen, J., Donehower, L., Lewis, D. E., Belmont, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+reporter+cell+line+for+HIV-1+tat+activity,+HIV-1+inhibitors,+and+T+cell+activation+effects.">A sensitive reporter cell line for HIV-1 tat activity, HIV-1 inhibitors, and T cell activation effects.</a> AIDS Res Hum Retroviruses. 10, 295-301 (1994).</li><li>Wu, Y., Beddall, M. H., Marsh, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rev-dependent+lentiviral+expression+vector.">Rev-dependent lentiviral expression vector.</a> Retrovirology. 4, 12-12 (2007).</li><li>Wu, Y., Beddall, M. H., Marsh, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rev-dependent+indicator+T+cell+line.">Rev-dependent indicator T cell line.</a> Current HIV Research. 5, 395-403 (2007).</li><li>Young, J., Tang, Z., Yu, Q., Yu, D., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+killing+of+HIV-1-positive+macrophages+and+T+Cells+by+the+Rev-dependent+lentivirus+carrying+anthrolysin+O+from+Bacillus+anthracis.">Selective killing of HIV-1-positive macrophages and T Cells by the Rev-dependent lentivirus carrying anthrolysin O from Bacillus anthracis.</a> Retrovirology. 5, 36-36 (2008).</li><li>Perez, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+HIV-1-infected+T+cell+clone+defective+in+IL-2+production+and+Ca2++mobilization+after+CD3+stimulation.">An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization after CD3 stimulation.</a> Journal of Immunology. 147, 3145-3148 (1991).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27, 493-497 (1938).</li></ol>

利害の衝突は宣言されません。

以前開発されたTAT -依存性の発現ベクターと同様に、ここで説明するレブシステムは進化HIVプロセスの開発です。 REV -依存性を含めると、複製HIVの存在に大きく依存してLTRベースの発現ベクターをレンダリングします。ここに報告されたとして、このベクトルの応用、HIV -依存性レポーターのウイルスは、HIV陽性細胞を同定するために斬新なアプローチを提供しています。生きた細胞のベクトルの許可検査では、基本的または臨床的実験のための任意の遺伝子を発現し、擬似型付けされたレンチウイルスとして最も細胞の種類や組織へのアクセス権を持つことができる。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Biosafety Cabinet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&deg;C 5%CO2 incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>FACSCalibur</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>Allega&trade;6KR</td>
<td>&nbsp;</td>
<tr>
<td>4&deg;C regrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>-20&deg;C refrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> -80&deg;C refrigerator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell counter</td>
<td>&nbsp;</td>
<td>Nexcelom</td>
<td>Cellometer&trade; AutoT4</td>
<td>&nbsp;</td>
<tr>
<td>HEK293T cell line</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CEM-SS cell line</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Jurkat cell line J1.1</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-118</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21870</td>
<td>&nbsp;</td>
<tr>
<td>HI FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438-018</td>
<td>&nbsp;</td>
<tr>
<td>HIV-1NL4-3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>prepared by tranfsection of HeLa cells with cloned proviral DNA</td>
</tr>
<tr>
<td>pNL-GFP-RRE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>copmlete deletion of all HIV ORFs of pNL4-3</td>
</tr>
<tr>
<td>pNL-GFP-RRE-SA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Insert HIV-1 A5 and D4 into pNL-GFP-RRE</td>
</tr>
<tr>
<td>pCMV&Delta;8.2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>provided by Dr. Dider Trono</td>
</tr>
<tr>
<td>pHCMV-G</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 x HBS (Hepes Buffered Saline)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11344-041</td>
<td>&nbsp;</td>
<tr>
<td>2M CaCl2 buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-013-154-10</td>
<td>&nbsp;</td>
<tr>
<td>1% paraformaldehy</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>158127</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50ml Falcon tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>358206</td>
<td>&nbsp;</td>
<tr>
<td>5ml round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352063</td>
<td>&nbsp;</td>
<tr>
<td>0.45&mu;M Millipore filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SLHA033SS</td>
<td>&nbsp;</td>
<tr>
<td>0.20&mu;M Millipore filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SLFG85000</td>
<td>&nbsp;</td>
<tr>
<td>100mm Petri-dish</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>CLS430588</td>
<td>&nbsp;</td>
<tr>
<td>Size-exclusion column (100,000MW cut off)</td>
<td>&nbsp;</td>
<td>SartoriuStedim biotech</td>
<td>VS2041</td>
<td>&nbsp;</td>
<tr>
<td>2ml frozen tube</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>SCT-200</td>
<td>&nbsp;</td>
<tr>
<td>96-well plate</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>353227</td>
<td>&nbsp;</td>
<tr>
<td>1.7mL Microcentrifuge Tube</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT- 175-C</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

specific marking of hiv-1 positive cells using a rev-dependent lentiviral vector expressing the green fluorescent protein

HIV -応答発現ベクターのほとんどは、HIVのプロモーター、長い末端反復（LTR）に基づいています。初期のHIVタンパク質に応答しながら、タット、LTRはまた、細胞の活性化状態へと統合が行われたローカルクロマチンの活性に応答する。これは、高いHIV -自主的な活動につながることができる、そしてHIV陽性細胞1,2,3をマークするLTRベースのレポーターの有用性が制限されています。ここで、我々は、タット応答LTR、REV -応答エレメントとHIVのスプライシング部位4,5,6を含む多数のHIVのDNA配列に加えて、所有している発現レンチウイルスベクターを構築した。ベクトルは、生きている細胞集団における複製HIVの非常に特異的な検出を可能にして、レンチウイルスレポーターのウイルスに組み込まれた。ベクトルの活動は、緑色蛍光タンパク質（GFP）の発現を測定した。ここに報告されたとして、このベクトルのアプリケーションは、HIV陽性細胞を識別するために、そのような現場 PCRやHIV抗原染色のような既存の方法、に新たな別のアプローチを提供しています。ベクターはまた、HIV陽性の細胞を標的とする基本的または臨床実験のために治療遺伝子を発現することができる。

Watch this Scientific Journal Video about Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein at JoVE.com

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

我々はTAT -応答LTR、HIV - 1 TAT -とRev依存的にレポーター遺伝子の発現を調節することができるREV -応答エレメント（RRE）に加えて、所有しているレンチウイルスベクターを開発した。ベクトルは、GFPの発現を介して、生細胞における複製HIVの特異的検出を可能にする。

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, ...

specific-marking-hiv-1-positive-cells-using-rev-dependent-lentiviral

Research

JoVE Journal

Immunology and Infection

12.8K ビュー.  George Mason University. 我々はTAT -応答LTR、HIV - 1 TAT -とRev依存的にレポーター遺伝子の発現を調節することができるREV -応答エレメント（RRE）に加えて、所有しているレンチウイルスベクターを開発した。ベクトルは、GFPの発現を介して、生細胞における複製HIVの特異的検出を可能にする。

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

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 ...

免疫・感染学

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 ...

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. ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

HIV-1感染細胞からのエキソソームのSILACベースのプロテオミクス特性評価

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

緑色蛍光タンパク質-表現する拡張を使用するマウス腹腔マクロファージの貪食能を評価するために大腸菌

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

REV免疫沈殿と質量分析によるHIV-1複製中のヌクレオール因子の同定

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...