작품은 DP2 OD004631와 R01 AI084119 포함한 빌 앤드 멜린다 게이츠 재단 (# 38631)와 NIH에서 교부금에 의해 지원되었다.

1. 방법론 <ol><li> 프로토콜의 첫 번째 단계는 silico에 접어하고자하는 V3 왕관 시퀀스를 선택합니다. R2 스트레인 들어,이 조각에 대한 시퀀스 KSIPMGPGRAFYT입니다. </li><li> 이 순서에 해당하는 펩타이드의 3D 원자 구조는 컴퓨터의 가상 공간에 건설되어야합니다. 이에 대한 ICM 명령은 다음과 같습니다 : buildpep &quot;KSIPMGPGRAFYT&quot; 또는 파일 : 새 : 펩타이드는 풀다운 메뉴에 따라 선택할 수 있습니다 </li><li> 절차의 몇 가지 매개 변수는 다음 검색 단계의 번호 (검색 길이), 시뮬레이션 온도, 바이어스 합리적인 지역 에너지 용어, 다른 에너지 계산 방법의 선택으로 검색을하기 위해 변수의 구속을 포함하여 검색 전략 매개 변수를 포함하여, 설정 그리고 검색이 같은 동영상을 기록 여부로 기록하고 얼마나 많은 중간 점수 conformations되는 방법을 나타내는 매개 변수가 기록되어야합니다. 이러한 모든 매개 변수는 이전의 출판물 (www.molsoft.com 참조)에 의해 최적화되었습니다. </li><li> 접지는 다음 명령으로 시작됩니다 : 몬테 칼 또는 파일 : 새 : 펩타이드는 메뉴 분자 역학 풀다운에 따라 선택할 수 있습니다 : 최소화 : 글로벌 전자의 경우에는 3 단계의 매개 변수가 명령줄에 하나씩 설정해야합니다. 명령이 실행되기 전에 후자의 경우, 가장 일반적으로 선택한 매개 변수에 대한 확인란이 패널에 제공됩니다. 편의를 위해,이 실험 이전에 출판 실험 활용 같은 접이식 ICM 스크립트는 여기에 나와 있습니다 : <img src="/files/ftp_upload/2118/2118output.jpg" alt="출력" /> 이 스크립트는 텍스트 파일에 저장하고 빠르게 명령을 사용하여 컴퓨터의 운영 체제 커맨드 라인 (일반적으로 리눅스)에서 실행할 수 있습니다 : ICM _foldingscript </li></ol> 2. 성공의 비밀 <ol><li> V3 루프는 거의 모든 알려진 변종의 길이는 상수 35 아미노산이며, 아미노산 위치가 1 시스틴 보세 원래 이황화 시작하여 35 시스틴 보세 해당 이황화과 종료 1에서 35 번호되도록. 그것이 루프의 일부이기 때문에, V3의 왕관의 경계를 신중하게 선택하여야한다. 너무 큰 조각을 선택한 경우는 무료 펩타이드 것처럼, 그것이 자유롭게 유연한 세그먼트로 동작하지 않을 수 있습니다, 접는 시뮬레이션이 제대로 구조를 평가되지 않습니다. 너무 작은 조각이 선택되면, 정보를 차 구조는 시뮬레이션에서 양식하지 않을 수 있습니다. 우리의 이전 연구는 위치 22 위치 10 조각은 항체 바운드 crystallographic conformations와 상관 것으로 나타났다, 그래서 이것은 접는 위해 선택되어야하는 V3 루프의 조각이다. </li><li> 그래픽 사용자 인터페이스를 사용하고 풀다운 메뉴는 사용자 친화이지만, 특히 ICM과 V3 루프 왕관 접는를 사용하여 일반적으로 펩타이드 접는에 이전 성공적인 작품은 단순히 buildpep 라인의 순서를 수정하고 실행 위의 스크립트를 사용하여 커맨드 라인에서 위의 스크립트를 권장합니다. </li></ol> 3. 대표 결과 R2의 접는에 대한 결과는 V3 루프에 대한 결과의 대표입니다. 결과를 평가하기 위해, 프로젝트 파일 (이것은 위의 스크립트에서 &quot;newProject1.icb&quot;라는 것입니다) 선택 &quot;을 분자 역학, 스택,보기&quot;개설하고 있어야합니다. 스택 conformations의 테이블이 나타납니다. 스택 conformations는 플롯 / 히스토그램 아이콘을 클릭하여 그래픽 시각하실 수 있습니다. &quot;분자 역학, 스택, 재생&quot;시각 접는에 의해 밝혀 conformational 환경 설정을 감사하는 스택의 영화를 만들 것입니다. R2 시퀀스의 경우 형태는 V3 루프 2 예상 베타 헤어핀 같은 것입니다 - 특히 위치에있는 조각에 12 깨끗한 β - 스트랜드 환경 설정이 스택에 걸쳐 볼 수 있으며 거의 알파 - 나선형 conformations입니다 14 볼 수 있습니다. 또한, 거의 3 단위의 에너지 격차가 가장 낮은 에너지 형태와 두 번째 낮은 에너지 형태 사이에서 볼 수 있습니다. 보기 활기 넘치는 관점에서,이 의미 낮은 에너지 형태의 구조는 깜박임 아웃 시간 미만 퍼센트 : 접는 결과는 따라서 R2 V3 왕관이 단단한보다는 유연한 구조로되어하는 것이 좋습니다. 이 conformations의 앙상블에 추가 중요한 구조적 기능을 수 있지만 그들은 체계적으로 감사하기가 어렵습니다 수 있습니다. <table border="1"><tbody><tr><td></td><td> JRFL </td><td> SF162_V3JRFL </td><td> SF162_V3R2 </td></tr><tr><td> 447 - 52D 그리니치 표준시 50 </td><td> 15 </td><td> 0.00061 </td><td> 0.00078 </td></tr></tbody></table> 표 1 : JRFL 및 R2 V3의 상대 중화가 가면과 가면 설정에서 루프. 데이터는 이전에 다음 ID로 카도조 T., 외. ARHR (200에서보고되었다8)와 핀터, A., 동부 표준시. 알 J. Virol (2004). SF162_V3JRFL 포함된 JRFL : 간단히, 중화 활동은 환경을 - 결함 루시페라제 - 표현 JRFL 환경을하거나 위에서 설명한 SF162 V3 변종과 함께 pseudotyped pNL4 - 3.Luc.RE - 플라스미드와 생성된 psVs을 사용하여 단일 사이클 감염 분석과 결정했습니다 SF162 V3 루프 시퀀스 대신에 V3 루프 시퀀스. SF162_V3R2는 SF162 V3 루프 시퀀스 대신에 R2의 V3 루프 시퀀스를 포함하고 있습니다. psVs는 37 1.5 H에 대한 447 - 52D mAb 일련 dilutions와 incubated했다 polybrene (10 MG = ML)의 면전에서 96 - 웰 플레이트에 도금 ° C, 그리고 CD4에 추가 + CCR5 + U87 대상 세포. 24 시간 후, 세포가 배양의 추가 24-48 H 다음 10% FBS 및 10 MG = ML polybrene을 포함하는 RPMI 매체와 refed되었습니다. 루시페라제 활동이 447 - 52D 50 %의 중화 (GMT50)에 대한 Promega (주) 기하 평균의 titers에서 분석 시약을 사용하여 microtiter 플레이트 luminometer (HARTA 주식 회사)와 48-72 H의 postinfection 결정되었고 중화 곡선의 보간에 의해 결정하고 있었다 적어도 세 독립 assays의 평균입니다.

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

관심 없음 충돌 선언하지 않습니다.

순이 론적 접힘은 차 구조 특성, 순차적으로 각각의 아미노산을 공부에서 뚜렷하지 않을 수있는 효과를 나타낸다. R2 V3 루프 왕관 몇 가지 이전에 애매한 구조 특성은 접는 시뮬레이션에 의해 공개됩니다. 이러한 관찰 구조적 환경 즉 R2 스트레인, 중화 및 hyperinfectivity 5 감도의 관찰 기능 특성과 상관 관계 수 있습니다. 첫째, 위치 12-14에서 R2의 V3 크라운의 N - ?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>ICM-Pro</td>
<td>&nbsp;</td>
<td>Molsoft LLC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

assessment of immunologically relevant dynamic tertiary structural features of the hiv-1 v3 loop crown r2 sequence by ab initio folding

HIV - 1의 antigenic 다양성 길이 백신 설계에 장애물을하고 있으며,이 변화는 특히 바이러스 &#39;표면 봉투 당단백질의 V3 루프의 발음입니다. 우리는 이전에 V3 루프의 왕관,하지만 역동적이고 순서 변수, immunologically 관련 β - 헤어핀 차 구조 HIV - 1 바이러스의 인구에 걸쳐 제약이 있다고 제안했습니다. 중요한 것은, 다른 V3 루프 왕관 시퀀스 수천 crystallography 나 NMR에 의해 어렵거나 불가능 바이러스의 다양성에 걸쳐 트렌드의 3D 구조 특성 만들기, HIV - 1 바이러스를 순환에 있습니다. V3 왕관 1, 2 접는과 함께 이전의 성공적인 연구 ICM - PRO 분자 모델링 소프트웨어 패키지 (Molsoft LLC, 라 호야, CA)에 접근 순이 론적 알고리즘 3 사용하고 제안하는 V3 루프의 왕관, 구체적으로부터 위치 10-22 충분한 유연성과 자유롭게 솔루션 접는 거리낌없는 펩타이드 것처럼 큰 정도로 동작하기 위해 측면 줄기의 길이에서 혜택을 제공합니다. 60000의 개별 변형의 V3 루프의 그냥이 부분 등, 빠른 순이 론적 접는로 + 순환 HIV - 1 계통 정보를 수 있습니다. 여기, 우리는 독특한 속성의 구조적 기초에 대한 통찰력을 얻으려면 R2 변형의 V3 루프를 접어. R2는 환자와 단클론 항체 세라 4 5 중화이 변형의 아름다운 감성에 대한 책임 것으로 생각 드문 V3 루프 시퀀스를 맺는다. 변형 CD4 독립 감염 mediates하고 널리 중화 항체를 이끌어내는 것으로 나타났습니다. 우리는 접는의 결과 평가가 R2에 대한 관찰 면역 활동과 접는에서 관찰 구조와 연결에 대한 정보를 수있는 방법을 보여줍니다.

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

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

HIV - 1의 표면 봉투 당단백질 (gp120)의 다른 V3 루프 시퀀스의 왕관 지역은 구조 위치의 silico 폴딩에 의해 대부분의 경우 특색 수있는 최첨단의를 사용하여 루프의 10-22<em&gt; 순이 론적</em&gt; 접는 알고리즘. 여기 우리는 HIV - 1의 R2 스트레인, 기괴 기능적 특성을 가진 독특한 중화 민감한 변형에서 V3 루프의이 지역의 접는 및 평가를 보여줍니다.

하여 HIV - 1 V3 루프 크라운 R2 시퀀스 Immunologically 관련 동적 차 구조 기능의 평가 순이 론적 접는

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

assessment-immunologically-relevant-dynamic-tertiary-structural

Research

JoVE Journal

Immunology and Infection

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

9.5K 조회수.  School of Medicine, New York University. HIV - 1의 표면 봉투 당단백질 (gp120)의 다른 V3 루프 시퀀스의 왕관 지역은 구조 위치의 silico 폴딩에 의해 대부분의 경우 특색 수있는 최첨단의를 사용하여 루프의 10-22 순이 론적 접는 알고리즘. 여기 우리는 HIV - 1의 R2 스트레인, 기괴 기능적 특성을 가진 독특한 중화 민감한 변형에서 V3 루프의이 지역의 접는 및 평가를 보여줍니다.

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

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

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

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, ...

연구

면역학 및 감염병학

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 ...

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

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

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

표준 임상 Assays에 의해 탐지의 한도 이하 바이러스성로드와 HIV 감염된 개인의 확장 및 HIV - 1 RNA를 Quantifying

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

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

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

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

Cryo - 전자 현미경에 의해 HIV - 1 Capsid 어셈블리의 구조와 반복 나선형 실제 공간 재건

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

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

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 ...

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.

뉴 클레오 캡시드 소둔 매개 전기 영동 (NAME) 분석은 HIV-1 뉴 클레오 캡시드 억제제의 신속한 식별을 할 수 있습니다

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 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. ...