著者らは、HIV - 1の多くの研究に参加した患者の認知を行う予定。 JMCは、FMカービー財団からの支援とアメリカ癌協会の研究教授を務めた。

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利害の衝突は宣言されません。

HIV - 1自然に感染を制御し、感染した抗レトロウイルス治療の個人（CART）または非常に低いウイルス負荷、プラズマ4、11、12、17、18のmlあたり、通常、約1コピーを持っている。自然なコントロール患者のウイルス負荷は、しばしば、個々のセットポイント1、2、17日頃変動する。ここに記載のアッセイの感度は、プラズマの入力量に大きく依存しています。一般的に、我々は?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ランダムヘキサマー（500 ug / mlと） </td><td>プロメガ</td><td> C118A </td><td></td></tr><tr><td>タックマンバッファ</td><td>アプライドバイオシステムズ</td><td> 4304441 </td><td></td></tr><tr><td> RNAsin（40 U / UL） </td><td>プロメガ</td><td> N211B </td><td></td></tr><tr><td> RT酵素（200 U / UL） </td><td> Strategene </td><td> 600107〜51 </td><td></td></tr><tr><td>のAmpliTaqゴールドのDNAポリメラーゼ</td><td>アプライドバイオシステムズ</td><td> 4311814 </td><td> 6パック</td></tr><tr><td>のAmpliTaq 10XGold PCR緩衝液II </td><td>アプライドバイオシステムズ</td><td> 4311814 </td><td>酵素が付属しています</td></tr><tr><td> Magnesiumcloride 25mMの</td><td>アプライドバイオシステムズ</td><td> 4311814 </td><td>酵素が付属しています</td></tr><tr><td> 1M Tris - HCl緩衝液、pH8.0、5mMの</td><td>インビトロジェン</td><td> AM9855G </td><td></td></tr><tr><td>上付き文字III逆転写酵素、200 U / UL </td><td>インビトロジェン</td><td> 18080-044 </td><td></td></tr><tr><td>上付き文字III 10 ×バッファー</td><td>インビトロジェン</td><td></td><td>酵素が付属しています</td></tr><tr><td> DTT 100mMの</td><td>インビトロジェン</td><td></td><td>酵素が付属しています</td></tr><tr><td>プラチナTaq DNAポリメラーゼハイフィデリティ5 U / UL </td><td>インビトロジェン</td><td> 11304-102 </td><td></td></tr><tr><td> 10XハイフィデリティPCRバッファー</td><td>インビトロジェン</td><td> 11304-102 </td><td>酵素が付属しています</td></tr><tr><td>プロテイナーゼK 20 mg / mlの</td><td>アンビオン</td><td> 2546 </td><td></td></tr><tr><td>グアニジニウムチオシアネート溶液を6M </td><td> FlukaBiochemica </td><td> 50983 </td><td></td></tr><tr><td>グリコーゲン20 mg / mlの</td><td>ロッシュ</td><td> 10901393001 </td><td></td></tr><tr><td>イソプロパノール</td><td>シグマアルドリッチ</td><td> 19516 </td><td></td></tr><tr><td> 70％エタノール</td><td>シグマアルドリッチ</td><td> E702 - 3 </td><td></td></tr><tr><td>分子グレードの水</td><td> GIBCO / Invitrogen社</td><td> 10977-015 </td><td></td></tr><tr><td>のdNTP（25ミリモル各） </td><td> Bioline </td><td> BIO - 39025 </td><td></td></tr></tbody></table><table border="1"><tbody><tr><td> Euipmentの名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>簡単なシールの遠心管（16x73mm） </td><td>シートンサイエンティフィック</td><td> 6041 </td><td></td></tr><tr><td>ホワイトデルリン冠</td><td>シートンサイエンティフィック</td><td> 4020 </td><td>簡単なシールチューブ用キャップ</td></tr><tr><td> Optisealベルトップチューブ、8.9 mlのチューブのラック</td><td>ベックマン</td><td> 361642 </td><td></td></tr><tr><td>六角ドライバー½株式会社の開口部</td><td>シートンサイエンティフィック</td><td> 4013 </td><td></td></tr><tr><td>キャップ除去tupe </td><td>シートンサイエンティフィック</td><td> 4014 </td><td></td></tr><tr><td> 5ミリリットル血清ピペット</td><td>共演スター</td><td> 4051 </td><td></td></tr><tr><td> Pipetboy </td><td>あらゆる</td><td></td><td></td></tr><tr><td> 15 mlの転送ピペット</td><td> ISC Bioexpress </td><td> P - 5005から7 </td><td></td></tr><tr><td> 5.8ミリリットルDispossable転送ピペット、ファインチップ</td><td> VWR </td><td> 14670-329 </td><td></td></tr><tr><td> 15ミリリットル遠心チューブ</td><td>ファルコン</td><td></td><td></td></tr><tr><td> 1 mlの遠心管</td><td>あらゆる</td><td></td><td></td></tr><tr><td>トリス緩衝生理食塩水の錠剤</td><td>シグマアルドリッチ</td><td> T5030 - 100錠</td><td></td></tr><tr><td>超遠心機とローター</td><td> Sorval </td><td> 90SEとT - 1270 </td><td></td></tr><tr><td> 96ウェルPCRプレート</td><td>バイオラッド</td><td> HSS - 9601 </td><td></td></tr><tr><td> 50 mlの試薬リザーバー</td><td>共演スター</td><td> 4870 </td><td></td></tr><tr><td>マイクロアンペア光学96ウェル反応プレート</td><td>アプライドバイオシステムズ</td><td> N801 - 0560 </td><td></td></tr><tr><td>光学プレートカバー</td><td>アプライドバイオシステムズ</td><td> 4333183 </td><td></td></tr><tr><td>マイクロアンペア光圧縮パッド</td><td>アプライドバイオシステムズ</td><td> 4312639 </td><td></td></tr><tr><td>マイクロ薄膜</td><td>バイオラッド</td><td> MSA - 5001 </td><td></td></tr><tr><td> 2 mlの遠心管</td><td>任意の</td><td></td><td></td></tr><tr><td> 1％アガロースゲル（E -ゲル、Invitrogen社） </td><td>インビトロジェン</td><td> G - 7008〜01 </td><td></td></tr><tr><td> E -ベース（Invitrogen社） </td><td>インビトロジェン</td><td> EB - M03 </td><td></td></tr></tbody></table> EDTA採血管の任意のブランド

amplifying and quantifying hiv-1 rna in hiv infected individuals with viral loads below the limit of detection by standard clinical assays

ウイルス遺伝子を増幅し、標準的なアッセイによる検出の限界（下の50〜75コピー/ ml）以下のウイルス量とHIV - 1感染個体においてHIV - 1 RNAを定量する患者でウイルスの動態とウイルスの宿主の相互作用への洞察を得るために必要がある人自然に感染し、抗レトロウイルス併用療法（CART）にある人々を制御する。 ここでは、単一のゲノムシークエンシング（SGSプロトコル）13、19とどのように正確に、低ウイルス量（シングルコピーアッセイ（SCA）プロトコル）12、20の患者でHIV - 1 RNAを定量化することによりウイルスゲノムを増幅する方法について説明します。 シングルコピーのアッセイは、アッセイされる血漿の量に応じて感度のリアルタイムPCRアッセイである。単一のウイルスのゲノムはプラズマ7mlに検出された場合、その後のRNAコピー数が0.3コピー/ mLと報告されている。アッセイは、RNAの抽出、およびDNAや汚染から可能な限り増幅するためのコントロールの効率化のための内部統制のテストを持っています。患者試料は三連で測定されます。 単一ゲノムシークエンシングアッセイ（SGS）、現在広く使用されていると、非労働集約的な3、7、12、14、15とみなさは、エンドポイントは、cDNA産物を96ウェルに分散している希釈した、限界希釈アッセイです。プレート。ポアソン分布によると、井戸の1 / 3以下の生成物を得たときに、単一のcDNA分子から増幅のPCR産物という結果の80％のチャンスがある。 SGSは、リサンプリングにさらされていないとPCR -導入組換え19によってバイアスされていないのクローニング利点があります。しかし、SCAおよびSGSの増幅の成功は、プライマーの設計に依存する。両方のアッセイはHIV - 1サブタイプBのために開発されましたが、変更のプライマー、プローブ、および基準で他の亜型とゲノムの他の地域に適合させることができる。

Watch this Scientific Journal Video about Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays at JoVE.com

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

検出限界（50〜75コピー/ ml）以下のウイルス量の個体から血漿とクエンシングにおけるHIV - 1 RNAのレベルを定量シングルHIV - 1ゲノムは困難です。ここでは、確実に0.3コピー/ mLにHIV - 1 RNAを測定すると非常に低いウイルス量の試料から、単一のゲノムの配列決定によってウイルスゲノムを増幅する方法を、リアルタイムPCRアッセイを用いて血漿中ウイルスRNAを抽出し、定量化する方法について説明します。

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 ...

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Immunology and Infection

31.3K ビュー.  NCI-Frederick. 検出限界（50〜75コピー/ ml）以下のウイルス量の個体から血漿とクエンシングにおけるHIV - 1 RNAのレベルを定量シングルHIV - 1ゲノムは困難です。ここでは、確実に0.3コピー/ mLにHIV - 1 RNAを測定すると非常に低いウイルス量の試料から、単一のゲノムの配列決定によってウイルスゲノムを増幅する方法を、リアルタイムPCRアッセイを用いて血漿中ウイルスRNA...

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

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

ナチュラルキラー細胞傷害性アッセイにおけるHIV - 1感染プライマリCD4 + T細胞を標的細胞としての準備と使用

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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 ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

HIV-1に感染した個体からの制御性T細胞を増殖するための新しいツール

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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 ...

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. ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

HIV-1に感染したライブ初代ヒトマクロファージで測定ファゴソームの移行と速度

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

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.