작품은 NIAID에서 YW에 보건 서비스를 부여 1R01AI081568로와 M. 로젠과 Day2 주식 회사가 주최하는 2009 NYCDC 에이즈 라이드의 기증자와 라이더의 관대한 기부금에 의해 부분적으로 지원되었다

1. 레브 의존 Lentiviral 입자의 생산을위한 Plasmids의 Transfection 설정 : 레브 종속 GFP lentiviral 벡터, pNL - GFP - RRE - SA는, 이전 4,5,6 설명했다. 바이러스 입자로 조립하려면, 플라스미드는 HIV - 1 포장 구조, pCMVΔ8.2 (친절 박사 Dider Trono에서 제공)와 HEK293 T 세포에 contransfected 및 플라스미드는 VSV - G의 당단백질 (pHCMV - G)를 가지고 가던 중이었습니다 . transfection은 인산 칼슘 방법을 사용하여 실시되었다. 버퍼의 준비 : 10 X HBS는 (Hepes가 살린 버퍼) Hepes 5 G를 해소하여 준비했습니다 8 NaCl의 g, 100 ML에 나 2 HPO 4 (무수)의 KCl, 덱스 트로 오스의 1g, 0.103 g의 0.37 g H 2 O. 나누어지는 the10 X HBS의 한 ML의 aliquots에 버퍼와 -20 ° C.에 저장 transfection의 당시 H 2 O (1 : 5 희석) 2 X HBS에 10 X HBS로 변환하고, 사이에 7.05로 산도를 조정 - 7.12. (10 ML 2 X HBS를 위해, 그것은 보통 1M NaOH의 약 50 μL가 필요합니다.) 0.22 μm의 Millipore 필터를 통과하여 2X HBS 버퍼를 소독 필터. CaCl 2 버퍼는 최종 농도 2M 10 MM의 Hepes에 CaCl 2를 용해하여 만들어졌다. 5.8으로 산도를 조절, 4에서 버퍼와 저장소를 소독 필터 ° C.를 절차 : <ol><li> 이전 transfection 한 일, 종자 2 X 10 6 HEK293T 페트리 - 요리 100mm의 세포, 37 10 ML DMEM + 10% FBS에서 야간 세포를 성장 ° C, 5 % CO 2. </li><li> 그 다음날, 세포의 표면에 뜨는 제거 5 ML 신선한 DMEM + 10% FBS, 4 시간 동안 지속적으로 문화 전지로 교체하십시오. </li><li> 5 ML의 폴리스티렌 관에서 480 μL 2X HBS 버퍼를 추가합니다. </li><li> 두 번째 5 ML의 폴리스티렌 튜브에 480 μL의 총 부피 60 μL 2M CaCl 2 버퍼, 플라스미드 DNA 및 transfection TE 버퍼를 (1 MM 트리스, 25 MM EDTA (에틸렌 다이아 민 테트라 초산), 산도 8.0)을 추가합니다. 각 페트리 - 요리, plasmids은 pNL - GFP - RRE - SA, pCMVΔR8.3의 7.5 μg, 그리고 pHCMV - G 2.5 MG 10 μg의 비율에 추가되어야합니다. </li><li> 2 X HBS 튜브에 dropwise 플라스미드 DNA - CaCl 2 혼합물을 추가하고 30 분 동안 상온에서 알을 품다. 좋은 침전물이 형성됩니다. </li><li> 세포에 피펫으로 dropwise 혼합물의 960 μL를 추가합니다. ° C, 5 % CO 2 밤새 37 알을 품다. </li><li> 다음날, 뜨는을 제거하고 10 ML 신선한 DMEM + 10% FBS를 추가하고, 지속적으로 37 ° C, 5 % CO 2 밤새에서 세포를 품어. </li><li> 48 시간 게시 transfection에서 수확 뜨는을 제거 50 ML 멸균 튜브에 전송하여 바이러스. 각 페트리 - 그릇에 신선한 10 ML 신선한 DMEM + 10% FBS를 추가하고 야간 문화를 계속합니다. 4 바이러스 뜨는을 저장 ° C. </li><li> 뜨는를 수집하여 72시간 게시물 transfection에 수확을 계속합니다. 모든 supernatants를 결합하고, 펠릿 15 분 500 XG에서 supernatants를 원심과 세포 잔해를 제거합니다. </li><li> 뜨는은 0.22 μm의 Millipore 필터를 통해 수집 및 필터링되었습니다. 바이러스는 더 크기 배제 컬럼을 통해 집중되었다. </li></ol> 2. 바이러스성 입자를 집중 바이러스성 Titer 결정 <ol><li> 바이러스성 입자는 크기 제외 열 (100,000 MW가 잘려, Centricoin)에 의해 집중되었다. 바이러스성 뜨는은 열에로드, 4 15 분 동안 ° C에서 6,000 XG에 centrifuged했다. </li><li> 집중 바이러스 뜨는가 수집 aliquoted하고, -80 ° C.에 저장된 </li><li> 바이러스 titer는 TNF - 처리, HIV - 1 양성 재킷 세포 라인, J1.1 7 감염에 의해 결정되었다. 전지는 ML 당 2.5 X 10 5 셀 (100 ML 전체 볼륨)에서 96 접시에 잘 교양하고, 일주일 동안 순차적으로 희석 vNL - GFP - RRE - SA 100 ML에 감염되었습니다. 입자의 titer (TCID50)는 리드와 Muench 8 방법 다음과 같은 GFP 긍정적인 우물을 계산하여 추정되었다. </li></ol> 3. 마킹 HIV - 1 Lentiviral 입자와 함께 긍정적인 세포, vNL - GFP - RRE - SA 설정 : 인간의 CD4 T 세포 라인, CEM - SS는, 먼저 HIV - 1에 감염되었습니다. 48 시간 이후 감염에서, 세포는 lentiviral 입자 vNL - GFP - RRE - SA와 함께 superinfected되었습니다. 또 이틀 동안 superinfection 따라 GFP 양성 세포 유동세포계측법에 의해 분석되었다. 컨트롤로서, HIV - 1 감염되지 않은 CEM - SS 세포는 동일 vNL - GFP - RRE - SA에 감염되었습니다. 단지 HIV - 1에 감염된 CEM - SS 세포는 HIV - 1 감염되지 않은 CEM - SS 세포를 GFP 양성 세포에 상승했다지만. 절차 : <ol><li> 감염 전에 두 시간은 최종 농도 4 MG / ML에 polybrane를 추가합니다. 세포를 카운트하고 각 감염 2 × 10 5 세포를 가져가라. 2 시간 동안 HIV - 1 NL4 - 3 200 NG (p24)의 세포를 감염. </li><li> resuspend은 10 분 300 XG에서 원심 분리기에 의해 세포를 세척48 시간 37 2 X 10 5 세포 / ML, 문화에 세포 ° C. </li><li> 세포를 카운트하고 vNL - GFP - RRE - SA와 함께 감염 당 2 × 10 5 세포를 가져가라. ° C 야간 37 vNL - GFP - RRE - SA와 부화 100 ML을 추가합니다. 세포를 씻어 2 × 10 5 세포 / ML에 resuspend. </li><li> 48 시간 게시 superinfection에서 유동세포계측법 분석을 수행합니다. 감염된 세포는 pelleted 20 분 실온에서 incubated 500 ML 1 % paraformaldehede로 resuspended되었고, 다음 FACScan 분석기 (백톤 디컨슨)에서 분석했다. </li></ol> 4. 대표 결과 실험이 성공적으로 수행하는 경우에는 컨트롤에 GFP 양성 세포가 HIV - 1 감염되지 않은 CEM - SS에서 검색되지 않습니다, 반면, 상당한 GFP의 인구는, 흐름 cytometer에 의해 HIV - 1에 감염된 CEM - SS 세포에 감지됩니다 세포. 실험이 성공적으로 수행하는 경우, 레브 의존 lentiviral 벡터, vNL - GFP - RRE - SA는 녹색 형광 단백질 (GFP) 표현의 측정을 통해 세포 인구 생활의 복제 HIV의 매우 구체적인 검출을 허용합니다. 이 예제에서 수확 CEM - SS 세포는 마우스 CD24, HSA에 대한 PE - 라벨 쥐의 단클론 항체와 스테인드되었고, 다음 HSA 및 GFP의 표현 모두에 대해 흐름 cytometer에 분석했다. <img src="/files/ftp_upload/2198/2198fig1.jpg" alt="그림 1" /> 그림 1. 여기 같이, 상당한 GFP의 인구가 vNL - GFP - RRE - SA (그림 1C)와 superinfected HIV - 1에 감염된 세포에서 발견되었다. 대조적으로, GFP 양성 세포는 lentiviral superinfection (그림 1A) 또는 HIV - 1 lentiviral superinfection (그림 1B)와 감염되지 않은 세포.없이도 HIV - 1 감염되지 않은 CEM - SS 세포에서 발견되지 않았습니다

<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>

관심 없음 충돌 선언하지 않습니다.

이전 개발 태트 종속적인 표현 벡터와 마찬가지로 수익 시스템은 진화 HIV 과정의 착취가 여기에 설명되어 있습니다. 레브 - 의존의 포함은 복제 HIV의 존재에 LTR 기반 발현 벡터가 매우 의존 렌더링. 여기보고로 벡터의 응용 프로그램 HIV에 의존 기자의 바이러스는 HIV - 양성 세포를 식별하는 소설 새로운 접근 방식을 제공합니다. 벡터는 살아있는 세포의 검사를 허용하는 기본 또는 임상 실험에 대한 유전자를 표현하고 의사 입력 lentivirus 같은 대부분의 세포 유형과 조직에 액세스할 수 있습니다 수 있습니다.

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		<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 - 응답 표현 벡터의 대부분은 HIV의 발기인, 긴 터미널 반복 (LTR)을 기반으로합니다. 초기 HIV 단백질, 태트 대응 반면, LTR는 세포 활성화 상태와 통합이 발생 지역 염색질 활동으로 반응합니다. 이것은 높은 HIV 독립적인 활동의 결과 수 있으며, 마르크 HIV 양성 세포가 1,2,3에 LTR 기반 기자의 유용성을 제한하고 있습니다. 여기, 우리는 태트 - 응답 LTR, 레브 - 반응 요소와 HIV의 splicing 사이트 4,5,6를 포함 수많은 HIV의 DNA 시퀀스 이외에, 가지고 표현 lentiviral 벡터를 잽니다. 벡터는 살아있는 세포 집단에서 HIV의 복제 매우 구체적인 감지를 허용, lentiviral 기자 바이러스에 통합되었다. 벡터의 활동은 녹색 형광 단백질 (GFP)의 표현에 의해 측정되었다. 여기보고로 벡터의 응용 프로그램은 HIV - 양성 세포를 확인하기 위해 현장 PCR이나 HIV의 항원 얼룩과 같이 기존의 방법에 새로운 대안의 접근 방식을 제공합니다. 벡터는 또한 HIV - 양성 세포를 대상으로 기본 또는 임상 실험을위한 치료 유전자를 표현할 수 있습니다.

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

우리는 태트 - 응답성 LTR 이외에, 가지고 lentiviral 벡터를 개발, HIV - 1 태트 및 레브 종속적인 방식으로 기자의 유전자 발현을 조절 할 수 레브 - 반응 요소 (RRE). 벡터는 GFP의 표현을 통해 살아있는 세포에서 HIV 복제의 구체적인 검색을 허용합니다.

그린 형광 단백질을 표현 레브 종속 Lentiviral 벡터를 사용하여 HIV - 1 양성 세포의 특정 마킹

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 조회수.  George Mason University. 우리는 태트 - 응답성 LTR 이외에, 가지고 lentiviral 벡터를 개발, HIV - 1 태트 및 레브 종속적인 방식으로 기자의 유전자 발현을 조절 할 수 레브 - 반응 요소 (RRE). 벡터는 GFP의 표현을 통해 살아있는 세포에서 HIV 복제의 구체적인 검색을 허용합니다.

비디오: 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.

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

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 ...

연구

면역학 및 감염병학

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]).

Genotypic 접근 방법을 사용하여 시퀀스 분석에 의한 HIV-1 Coreceptor 사용 (Tropism)의 예측

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 코어의 시험 관내 Uncoating에서

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 감염 CD4의 타고난 감지 평가<sup&gt; +</sup&gt;를 사용하여 형질 수지상 세포에 의한 T 세포<em&gt; 예 생체 내</em&gt; 공동 문화 시스템.

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.

셀 기반의 ELISA 분석을 사용하여 HIV-1 삼량 봉투 당 단백질의 구조적 평가

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 양성 개인의 플라즈마에서 엑소 좀의 분리

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-1에 감염된 세포에서 엑소 좀의 SILAC 기반 단백체 특성

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.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

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.

녹색 형광 단백질 표현 하는 향상 된 사용 하 여 대장균 마우스 복 막 대 식 세포 식 균 작용을 평가 하기 위해

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.

레브 면역 침전 및 질량 분광법을 통한 HIV-1 복제 중 핵균 요인 식별

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