These studies were funded by US Public Health Services grants P01AI057005, R01AI047734, R01AI111806 and the Functional Profiling and Computational Biology Core of the University of Washington&#8217;s Center for AIDS Research (P30 AI027757).

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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다. 표현 의견은 여기에 저자의 견해이며 공식 또는 미 국방부 또는 육군의 부서의 의견을 나타내는 것은 아니다.

재조합 HIV-1 분자 클론 및 성장 경쟁 분석의 건설 : 제시된 프로토콜은 두 가지 주요 부분으로 구성되어있다. 이중 감염된 세포 배양 물에서 두 바이러스와 구별하기 위해, 경쟁 분자 클론을 RT-qPCR에 프라이머 - 프로브 분석으로 또는 생거 시퀀싱에 의해 검출 될 수있는 서열 태그를 포함하는 것이 중요하다. 이 프로토콜은 HIV-1의 NL4-3 VIF 동일한 영역을 점유하고 동일한 아미노산 서열을 인코딩하지만 여섯 동의어 돌연변이 차이 VIFA 및 VIFB 태그를 사용한다. 이러한 돌연변이는 영향을주지 나타냈다 바이러스 복제의 적합성 (20). 이 프로토콜에서 사용하는 PCR 계 클로닝 방법은 제한 부위 기반 복제에 비해 클로닝 사이트를 선택하는데 더 많은 유연성을 제공한다. 그러나, PCR 클로닝의 효율은 인서트의 크기가 증가함에 따라 감소한다. 인서트 크기의 전류 제한은 ~ 5킬로바이트 (34)이다. PCR 기반의 복제 및 사이트 감독 mutagenesi 들어S, 고 충실도 DNA 중합 효소의 사용은 추가적인 돌연변이 가능성을 줄이기 위해 매우 중요하다. 제품의 충분한 양을 산출하기 위해 필요한 PCR 사이클의 최소 수의 사용은 권장된다. 우리의 경험에서, 우리는 여기에 설명 된 PCR 기반의 복제 및 부위 특이 적 변이 단계 후 여분의 돌연변이를 검출하지 않았다. 그럼에도 불구하고 PCR 제품은 원치 않는 돌연변이를 확인하기 위해 서열화한다. 이상적으로, 전체 HIV 코딩 영역을 resequenced해야합니다. 적어도 세 개의 샘플링 시점은 지수 적 성장 단계 9에서 검토되어야한다. 바이러스의 성장 동역학을 먼저 성장 경쟁을위한 적절한 배양 시간 및 샘플링 시간의 포인트를 결정하기 위해 매일 샘플링을 사용하여 설정되어야한다. 바이러스 성 성장 반응 속도에 영향을 미치는 하나의 요인은 감염 (MOI)의 다양성이다. 그것은 더 RO을 수득 하였다 나타낸 바와 같이이 프로토콜은, monoinfection 성장 경쟁 모두 0.005 총 MOI를 사용낮은 MOIs 9보다 흉상 결과. 그럼에도 불구하고, 낮은 MOIs 필요하다면 장기간의 지수 성장기를 얻기 위해 사용되지만, 결과의 일관성을 희생 할 수있다. 이 프로토콜은 바이러스의 체력 차이가 미리 일반적으로 알려지지 않은 것으로 가정하여, 50:50의 초기 감염 비율을 의미한다. 바이러스 복제 속도론에 상당한 차이점을 시사 예비 데이터가있는 경우에는, 동일하지 않은 입력 비율의 사용이 적합하다. 이러한 경우에는, 70:30의 비율은 감염 바이러스가 덜 맞는 초과 9에 배치 큰 차이 니스의 검출을 허용하도록 권장한다. 바이러스 성장 속도론 및 HIV-1 서브 타입 B를 사용하여 경쟁 분석 최적화 된 성장을 수행하기위한 TCID 50을 결정하기위한 프로토콜은, PBMC를 하나의 기증자로부터 NL4-3 COTB-P24 및 PBMC를이 HIV-하는 일관된 감수성을 증명하고있다 체외에서 1 감염. 배양 기간이 프로토콜에서 제공 샘플링 시점에 인간 PBMC를 HIV-1 바이러스 M 군을 연구에 적합한 것으로 보인다. 단일 소스에서 PBMC를 사용하는 것은 매우 바이러스 복제가 다른 공여 세포 (35)에 따라 다릅니다 수있는 일관성있는 결과를 얻을 것을 권장합니다. 교체는 동일한 풀 모든 실험에 걸쳐 사용되는 것을 규정 덜 바람직하지만, 복수의 공여체로부터 풀링 된 PBMC를 사용할 수있다. 또 다른 대안은 세포주의 사용이다. 여기에 제시된 프로토콜 T 세포주 CEMx174 23,36 성공적으로 사용되었다. 그러나, 세포의 파종 번호 재 최적화 일관된 세포 성장을 달성하는 것이 중요하다. 그것이 성장 경쟁 단계에 적절한 샘플링 시점을 결정하기 위해, 상이한 세포주에서 다를 가능성이 바이러스 성 성장 동역학은 또한 재 확립되어야한다. 체력을 계산하는 바이러스 성 비율을 결정하는 두 가지 방법이 protoc에 포함되어 있습니다OL. 첫째는 각 샘플링 시점에서 바이러스의 cDNA 카피 수를 측정하기 위해 RT-qPCR에 사용. 바이러스 복제 적당이어서 cDNA의 사본 번호에 기초하여, 바이러스의 비로부터 계산 하였다. 대안으로, 바이러스 성 비 VifAB 태그 사이트의 크로마토 그램의 피크 높이의 비율에 기초하여 결정될 수있다. 두 가지 방법이 유사한 결과 (도 4)을 수득 하였다. 크로마토 그램의 피크 높이 방법은 서열 태그 조작없이 다른 HIV-1 균주에 적용될 수있다. RT-qPCR의 바이러스 변이체를 구별하기위한 프라이머 또는 프로브의 사용은 제 신중하게 평가되어야 들어 (20 참조). 그럼에도 불구하고, RT-qPCR에 같은 지수 성장기 내의 제 시점에서 그와 같은 바이러스 성 RNA의 소량의 샘플과 더 나은 민감도를 제공한다. 바이러스 cDNA를 직접 측정은 또한 RNA 추출 및 cDNA 합성에서 발생할 수있는 기술적 인 문제를 감지 할 수 있습니다. 서열 태그 분자 클론을 사용하는 것은 비용 효율적인 솔루션을 제공 tRT-qPCR에 방법 O를, 같은 프라이머 및 프로브의 두 쌍은 여러 바이러스를 연구하기 위해 필요하다. 이 전략은 또한 시퀀싱이 연구에서 모든 바이러스에서 동일한 사이트에서 수행되는 것처럼 순서로 피크 높이 변화의 문제, 이웃 기지 (37)에 의한 크로마토 그램 방지 할 수 있습니다. RT-qPCR에와 생거 시퀀싱 생성 된 PCR 제품은 다른 방법과 함께 사용은 개별 클론 12, 13의 대량 순서, HTA 4,7,17,19, 또는 올리고 뉴클레오티드 결찰 분석 (OLA) (38) 바이러스 성 비율을 결정하기 위해 할 수 있습니다 .

바이러스 복제의 니스는 주어진 환경에서 한 감염성 자손을 생산하는 바이러스의 능력으로 정의하고 2 시간 이상 인구 수준에서 바이러스 변이체의 유병률을 결정하는 중요한 요인이다. 생체으로 피트니스 연구는 HIV-1과 같은 병원성 인간 바이러스, 다양한 vitro와 in와 가능하지 않은 생체 복제 피트니스 분석 약제 내성과 면역 탈출 돌연변이, 상위 성 및 진화에서 발생하는 체력에 미치는 영향을 연구하기 위해 개발되었다 바이러스 성 인구 3-6. 다른 피트니스 분석 중 성장 경쟁 분석은 생체 -1,7,8,8- 발생으로 두 개 이상의 바이러스 변종, 정확하게 동일한 환경 조건 하에서 동일한 세포 인구를위한 경쟁으로, 피트니스 차이에 더 민감하고 유효한 조치를 얻을에 인식하고 있습니다. 성장 경쟁 실험을 시작, 여러 variab 전레 감염의 다양한 다중성 (MOI), 바이러스의 입력 비율 및 분석을위한 샘플링 타이밍의 사용을 포함하여 결정되어야한다. 우리는 바이러스 성 성장 속도론에 경쟁 실험의 결과에 이러한 매개 변수의 효과를 공부하고, 세포 배양 9 HIV-1의 적합성에 강력한 측정에 필요한 핵심 요소를 확인했다. 분석 변수 외에도, 성장 경쟁 실험에서 바이러스 변이체를 정량을위한 다양한 방법이있다. 10,11 또는 벌크 클론 시퀀싱 12,13은 관심 부위 (들)에서 뉴클레오티드 주파수에 기초하여 상기 경쟁 바이러스의 비율을 결정하기 위해 사용되어왔다. 상대 체력은 시간이 지남에 따라이 비율의 변화에​​서 파생됩니다. DNA 시퀀싱 서비스를 광범위하게 사용할 수있는이 방법은 편리하다. 병렬 대립 유전자 특정 염기 서열 (PASS) 방법은 여러 사이트에서 시퀀싱 및 재조합 (14)의 검출을 가능하게 </sup&gt;뿐만 아니라 특별히 개발 시약 및 탐지 시스템을 필요로한다. 본질적으로,이 방법은 관심 영역의 뉴클레오티드 차이의 소수의 바이러스 균주를 개발하기 위해 연구 하였다. 다른 방법은 작은 리포터 유전자를 시퀀싱하여 경쟁 바이러스 heteroduplex를 추적 분석 (HTA) 4,7,17,19 구별 또는 전사 정량 실시간 중합 효소 연쇄 반응을 반대로 태그 15 또는 돌연변이 4,16-18 동의어를 사용 에 관계없이 서열 유사성의 균주 경쟁 연구에 적용 할 수 모두 (RT-qPCR에) 15,16,18,20. 추가 단계는 바이러스의 게놈에 태그를 도입하는 데 필요한 및 RT-qPCR에 분석은 특정 시약 및 장비를 필요로한다. 발명자들은 대량 생거 시퀀싱 수익률 비교 결과 (9). 성장 경쟁에 이어, 바이러스 복제 피트니스는 상대의 적합성, 또는 T 사이의 피트니스 비율로 제공됩니다바이러스 변종 하시다. 바이러스의 상대적인 니스는 접종의 초기 비율에 의해 또는 두 바이러스 간의 경쟁 순 성장 속도 차이로 규격화 바이러스 변이체의 최종 비율로 정의 할 수있다. 우리는 후자의 방법은, 단지 지수 성장기 내에 길이 데이터 포인트를 이용하여, 가장 견고한 결과 9,20 생산하였습니다. 체외에서 피트니스 분석은 생물학적 클론 6-8과 HIV-1 감염 분자 클론을 연구하기 위해 주로 사용된다. 후자는, 유전자 조작 의무가되고, 종종 특정 돌연변이 또는 관심 3-5,21,22의 특정 시퀀스에서 체력에 미치는 영향을 연구하기 위해 사용된다. 다음 프로토콜은 바이러스 성 성장 속도론을, 일련의 태그를 포함하는 HIV-1 벡터를 사용하여 새로운 전체 길이 감염성 HIV-1 분자 클론을 구성하는 관심의 돌연변이를 도입, 바이러스 성 주식을 만들고 구축의 관점에서 워크 플로를 설명상대 체력을 성장 경쟁 분석을 수행하고 계산하는 (그림 1). 우리의 최적화 된 절차를 사용하여, 우리는 세 가지 재조합 HIV-1 돌연변이를 만들어 자신의 복제 체력을 결정했다. 재조합 분자 복제가 먼저 합성 COTB (센터 외과, pNL4-3, HIV-1 실험실 변형 NL4-3의 전체 길이 감염 유전자를 포함하는 플라스미드의 HIV-1 개그-P24 유전자 영역을 대체하여 건설되었다 나무, 서브 타입 B) 개그-P24 시퀀스 (23)는 프로토 타입의 변형을 만들 수 있습니다. 단일 아미노산 변경 (T186M은 T242N 및 I256V는) 다음 세 가지 돌연변이 클론을 생성하기 위해 도입되었다. 각각의 돌연변이는 주어진 유전 적 배경에서 각 돌연변이의 체력에 미치는 영향을 관찰하기 위해 프로토 타입 바이러스에 대해 경쟁했다. 세 가지 돌연변이 프로토 타입 바이러스보다 약간 크게 낮은에서 복제 fsitness 다양한 수준의 보여 주었다. T242N 돌연변이는 이전에 적당한 fitne이보고되었다SS는이 연구에 나타낸 결과와 유사한 24-26, 비용. 다른 두 변이의 피트니스 비용은 이전에보고되지 않았다.

<table><tbody><tr><td>Construction of recombinant clones</td><td></td><td></td><td></td></tr><tr><td>Chimeric primer/Mutagenic/Sequencing primers</td><td>IDT</td><td></td><td>Custom DNA oligos</td></tr><tr><td>pNL4-3VifA and pNL4-3VifB plasmid</td><td></td><td></td><td>Please contact Dr. James I Mullins (jmullins@uw.edu)</td></tr><tr><td>High-Fidelity DNA polymerase</td><td>Thermo Scientific</td><td>F-549S</td><td></td></tr><tr><td>High-fidelity buffer</td><td>Thermo Scientific</td><td>F-549S</td><td></td></tr><tr><td>dNTP</td><td>Bioline</td><td>Bio-39026</td><td></td></tr><tr><td>Thermal cycler</td><td>Life Technologies</td><td></td><td>Applied Biosystems&amp;reg; GeneAmp&amp;reg; PCR System 9700</td></tr><tr><td>DNA loading dye</td><td>Thermo Scientific</td><td>R0631</td><td></td></tr><tr><td>1 kb Plus DNA ladder</td><td>Invitrogen</td><td>10787-018</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>QIAquick gel extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>DpnI enzyme</td><td>New England Biolabs</td><td>R0176S</td><td></td></tr><tr><td>TOP10 chemically competent &lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>Invitrogen</td><td>C4040-10</td><td></td></tr><tr><td>Luria Broth base powder</td><td>Invitrogen</td><td>12795-084</td><td></td></tr><tr><td>Carbenicillin</td><td>Research Products International</td><td>C46000-25.0</td><td></td></tr><tr><td>QIAprep Spin Miniprep kit&amp;nbsp;</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Transfection</td><td></td><td></td><td></td></tr><tr><td>X-tremeGENE 9 DNA transfection reagent</td><td>Roche</td><td>6365787001</td><td></td></tr><tr><td>HEK 293T-17</td><td>ATCC</td><td>CRL-11268</td><td>http://www.atcc.org/</td></tr><tr><td>0.22 &amp;#956;m filter top tube</td><td>VWR International</td><td>89220-716&amp;nbsp;</td><td></td></tr><tr><td>Cell culture</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>10566016</td><td></td></tr><tr><td>Iscove&amp;prime;s Modified Dulbecco&amp;prime;s Medium (IMDM)</td><td>Life Technologies</td><td>31980-030</td><td></td></tr><tr><td>RPMI-1640 media</td><td>Life Technologies</td><td>61870-036</td><td></td></tr><tr><td>Phytohemagglutinin (PHA)</td><td>Thermo Scientific</td><td>R30852801</td><td></td></tr><tr><td>Human Interleukin-2</td><td>Roche</td><td>11147528001</td><td></td></tr><tr><td>Fetal bovine serum</td><td>JR Scientific</td><td>43640</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>Corning Cellgro</td><td>30-001-CI</td><td></td></tr><tr><td>6 well plate</td><td>VWR Scientific</td><td>73520-906</td><td></td></tr><tr><td>48 well plate</td><td>VWR Scientific</td><td>62407-338</td><td></td></tr><tr><td>96 well flat-bottomed plate</td><td>ISC Bioexpress</td><td>T-3015-4</td><td></td></tr><tr><td>96 well round-bottomed plate</td><td>BD Falcon</td><td>353077</td><td></td></tr><tr><td>1.5 ml Microcentrifuge Tube</td><td>Mt. Baker Bio</td><td>MBD-1500</td><td></td></tr><tr><td>1.5 ml tubes w/ O-ring</td><td>VWR Scientific</td><td>89004-290</td><td></td></tr><tr><td>50 ml conical tube</td><td>ISC Bioexpress</td><td>C-3317-6</td><td></td></tr><tr><td>ELISA</td><td></td><td></td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>Invitrogen</td><td>14190-250</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F0392</td><td></td></tr><tr><td>glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Rabbit anti-HIV-1 SF2 p24 antiserum</td><td>NIH AIDS Reagent Program</td><td>4250</td><td></td></tr><tr><td>Goat anti-rabbit HRP</td><td>KPL</td><td>474-1516</td><td></td></tr><tr><td>TMB Microwell Peroxidase Substrate</td><td>KPL</td><td>52-00-01</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>A300-500</td><td>Causes severe burns by all exposure routes. Use personal protective equipment. Use only under a chemical fume hood. Wash off immediately with plenty of water for at least 15 min.</td></tr><tr><td>HIV p24 standard</td><td>AIDS and Cancer Virus program (NCI-Frederick)</td><td></td><td>For order details please contact Julian Bess, Jr. (bessjw@mail.nih.gov); http://ncifrederick.cancer.gov/Programs/Science/Acvp/bio/Bess.aspx</td></tr><tr><td>Microplate reader</td><td>Molecular Devices</td><td></td><td>VMax Kinetic ELISA Microplate Reader</td></tr><tr><td>RNA isolation cDNA synthesis</td><td></td><td></td><td></td></tr><tr><td>QIAxtractor</td><td>Qiagen</td><td></td><td>http://www.qiagen.com/products/catalog/automated-solutions/sample-prep/qiaxtractor</td></tr><tr><td>QIAamp Viral RNA Mini Kit</td><td>Qiagen</td><td>52906</td><td></td></tr><tr><td>dNTP</td><td>Bioline</td><td>Bio-39026</td><td></td></tr><tr><td>SuperscriptIII</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>First-strand buffer&amp;nbsp;</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>Rnase inhibitor</td><td>Roche</td><td>3335402001</td><td></td></tr><tr><td>RnaseH</td><td>Invitrogen</td><td>18021-071</td><td></td></tr><tr><td>qPCR</td><td></td><td></td><td></td></tr><tr><td>Real-time qPCR machine</td><td>Life Technologies</td><td></td><td>Applied Biosystems 7300 Real-Time PCR&amp;nbsp;</td></tr><tr><td>TaqMan&amp;reg; Gene Expression Master Mix</td><td>Life Technologies</td><td>4369016</td><td></td></tr><tr><td>Forward, reverse primer</td><td>IDT</td><td></td><td>Custom order</td></tr><tr><td>Probe</td><td>Life Technologies</td><td></td><td>Custom order</td></tr><tr><td>optical 96 well reaction plate</td><td>Life Technologies</td><td>I19N3Q216</td><td></td></tr><tr><td>PCR+sequencing</td><td></td><td></td><td></td></tr><tr><td>Taq DNA polymerase (Biolase)</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>NH4 buffer&amp;nbsp;</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>Sequencing primer</td><td>IDT</td><td></td><td>Custom order</td></tr><tr><td>QIAxcel</td><td>Qiagen</td><td></td><td>http://www.qiagen.com/products/catalog/automated-solutions/detection-and-analysis/qiaxcel-advanced-system</td></tr><tr><td>DNA sequencing service provider</td><td>http://www.htseq.org/</td><td></td><td></td></tr><tr><td>GRC web tool</td><td>http://indra.mullins.microbiol.washington.edu/grc/</td><td></td><td></td></tr><tr><td>ChromatQuan web tool</td><td>http://indra.mullins.microbiol.washington.edu/cgi-bin/chromatquant.cgi</td><td></td><td></td></tr></tbody></table>

pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

HIV-1 개그-P24 단일 아미노산 변화의 체력에 미치는 영향을 연구하기 위해, 우리는 HIV-1 COTB-P24 유전자 23,32,33를 포함하는 pNL4-3 플라스미드에 돌연변이를 소개하는 올리고 뉴클레오티드 특이 적 변이를 사용했다. 바이러스 주식 293T 세포의 형질 감염에 의해 생성 된, 48 시간 후에 수거 하였다. 우리는 리드 - MUENCH 방법 (31)에 의해 각각의 바이러스 주식의 50 % 조직 배양 감염 용량 (TCID 50)을 추정했다. 프로토 타입의 TCID (50)와 돌연변이 바이러스는 104에서 105 IU / ㎖ (그림 3A)였다. 재조합 바이러스의 성장 반응 속도는 0.005의 MOI에서 단일 기증자 PBMC를 설립했다. RT-qPCR의 여섯 일 동안 매일 바이러스 cDNA의 사본 수를 측정 하였다. 모든 돌연변이 및 프로토 타입 바이러스는 &lt;바이러스 RNA의 카피 수 증가의 감소 기울기 (로 표시된 바와 같이 바이러스 성 성장이 둔화 하루 2, 4 일, 다음 사이에 기하 급수적으로 증가강한&gt; 그림 3b). cDNA의 감소는 (접종에 해당) 0 일 및 2 일 사이의 수는 세포에 바이러스의 흡수 하루 1 세척 (단계 5.1.6)에 의해 언 바운드 비리의 제거로 인한 복사합니다. 지수 적 성장 단계에서, 모든 세 돌연변이 바이러스 원형 (도 3c)보다 느린 성장 속도 (g)를 가졌다. 세 가지 돌연변이 0.005 총 MOI에서 성장 경쟁 분석에서 프로토 타입 바이러스에 대해 경쟁했다. 이중 감염된 문화 바이러스 성 성장 속도론은 monoinfection의 그것과 유사 하였다; 바이러스 성 지수 성장기는 2 일 및 4 사이이고, 바이러스 성장은 5 일 (도 4a)의 주위에 도달 고원. 바이러스 성장 속도의 차이는 시간에 따른 바이러스의 비율의 변화로부터 유래 하였다. 비 바이러스의 cDNA 복사본 참조 번호 및 RT-qPCR에를 사용하여 돌연변이 바이러스와 비교하여 피크의 높이에 의해 산출했다시퀀스에서 두 바이러스 (도 4B)를 구분하는 뉴클레오티드 부위에서 크로마토 그램. 피크 높이와 바이러스 RNA 카피 수의 방법을 이용하여 결정 성장 속도의 차이는 유사한 결과 (도 4C)을 수득 하였다. 세 가지 돌연변이가 가장 낮은 피트니스 (그림 4C)을 갖는 돌연변이 I256V와 프로토 타입 바이러스보다 낮은 복제 체력을 가지고 있었다. <img alt="그림 1" src="/files/ftp_upload/52610/52610fig1.jpg" /> 그림 1 : 본 논문에서 제시 한 프로토콜의 흐름도 바이러스 주식의 바이러스 준비 프로토콜 우려 HIV-1 재조합 클론의 건설 및 생성.. 피트니스 분석 실험 프로토콜은 바이러스 성 성장 속도론을 수립하고 관련 바이러스의 적합성을 결정하기위한 것입니다. 점선은 프로토콜에 대한 다른 흐름을 나타냅니다. 예를 들어, 돌연변이 HIV-1 NL4-3로 직접 도입 될 수있다재조합 복제를 생성하지 않고 분자 클론. <img alt="그림 2" src="/files/ftp_upload/52610/52610fig2.jpg" /> 그림 2 :. 오버랩 확장 PCR (A)를 사용하여 HIV-1 NL4-3 COTB 개그-P24 재조합 분자 클론의 건설 키메라 프라이머를 디자인합니다. 5 &#39;프라이머의 절반은 NL4-3 벡터 시퀀스를 포함하고 3&#39;반쪽 삽입 시퀀스의 끝을 포함한다. (B) 키메라 프라이머는 삽입 조각, COTB 개그-P24를 증폭하는 데 사용됩니다. (C) 삽입 단편은 재조합 플라스미드를 생성하기 위해 두 번째 PCR 용 프라이머로 사용된다. <img alt="그림 3" src="/files/ftp_upload/52610/52610fig3.jpg" /> 그림 3 :. PBMC를에서 바이러스 성 성장 특성 (A) 로그 (10) TCID (50) </sUB&gt; 바이러스 주식. (B) MOI = 0.005에서 시작 monoinfections에서 바이러스 성 성장 속도론. 패널 (B) 유래의 지수 성장기 (2-4 일) 등 6 일 (C) 바이러스 성장율. 도시 된 값들은 하나로부터 3 회 반복 실험의 평균을 나타낸다. 오차 막대는 95 % 신뢰 구간을 나타낸다. <img alt="그림 4" src="/files/ftp_upload/52610/52610fig4.jpg" /> 그림 4 : 바이러스 성 성장과 상대의 적합성 결정 (A) 이중으로 감염 PBMC 문화에서 바이러스 성 성장.. T242N 및 프로토 타입 바이러스 (B) 비는 RT-qPCR에 또는 시퀀싱 피크 높이 법을 사용하여 측정. (A)에 도시 된 바와 같이, (C) 및 이중 돌연변이 감염된 배양에서 프로토 타입 바이러스 간의 순수한 바이러스 성장율 차이 (d). D 값은 f를 계산했다(B)에 도시 된 비 바이러스 성 데이터를 ROM. 도시 된 값들은 하나로부터 3 회 반복 실험의 평균을 나타낸다. 오차 막대는 95 % 신뢰 구간을 나타낸다.

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Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

인간 면역 결핍 바이러스의 복제 피트니스를 결정하기위한 인접 쌍 성장 경쟁 분석

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

pairwise-growth-competition-assay-for-determining-replication-fitness

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.1K 조회수.  University of Washington. Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

비디오: Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

다음-세대 시퀀싱에 대 한 전체 길이 HIV-1 Proviruses 근처의 증폭

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

연구

유전학

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

CRISPR-Cas9-기반 경쟁 분석 실험을 사용 하 여 유전 종속성의 조사

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

암 연구학

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV 역전사 및 인테그라 제 억제제의 빠른 검사

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

면역학 및 감염병학

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

제한 효소 기반의 복제 방법을 평가하기 위해<em&gt; 시험관</em&gt; HIV-1 서브 타입 C 개그 - MJ4 키메라 바이러스의 복제 능력

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

유전자 또는 약물 치료에 사용하기 위해 HIV-1 생산을 표적 RNA를의 효능 및 독성 평가

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

측정<em&gt; 체외</em&gt; HIV-1 사전 통합 단지의 통합 활동

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

전 비보 인간 면역 결핍 바이러스 1와 Histoculture 인간 림프 조직 및 여성 생식 기 점 막의 감염

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

높은 처리량 Cre Lox 활성화 에이즈-1 바이러스 막 융해의 억제제를 식별 하기 위해 바이러스 막 융합 분석 결과

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites.
In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed.
By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

CRISPR-Cas9-기반 게놈 엔지니어링 선택한 Proviral 통합 사이트와 에이즈-1 감염에 대 한 Jurkat 기자 모델을 생성 하

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we ...