우리는 그의 관대 한 A32, 17B, 48D의 선물 및 C11 모노클로 날 항체 박사 제임스 로빈슨 감사합니다. PGT (121)는 NIH AIDS 시약 프로그램, AIDS, NIAID, NIH (고양이 # 12343) 부문을 통해 얻었다. 이 작품은 과학자 AF와 CRCHUM 연속체 보조금에 의해뿐만 아니라 CIHR 촉매 부여 # 126630로 # 24639을 부여 영 FRQS 설립하여, CIHR 운영 # 257792로, 혁신 프로그램 리더 # 29866에 대한 캐나다 재단에 의해 지원되었다 AF 및 MR합니다. AF는 FRSQ Chercheur Boursier 주니어 1 교제 번호에 24639를받는 사람이다. MV는 CIHR 박사 연구 상 번호 291485에 의해 지원되었다.

1 일 한 - 세포 배양 <ol><li> 플레이트 2 × 10 4 인간 골육종 (HOS) 발광 읽기에 적합한 불투명, 96 웰 세포 배양 접시에 잘 당 세포. 10 % 소 태아 혈청 (FBS) 및 100 U / ㎖ 페니실린 - 스트렙토 마이신이 보충 된 둘 베코 변형 이글 중간 (DMEM)를 사용합니다. 37 ° C, 5 % CO 2에서 다음 날까지 품어. </li></ol> 2 2 일 - 폴리에틸렌 이민 (PEI) 형질 <ol><li> 다음 단계에 따라 형질 믹스를 준비합니다. 동일한 봉투 형질하여야 웰의 수에 따른 시약 및 DNA의 양을 조정한다. </li><li> 튜브는 25 mM의 HEPES와 보충 5 μL의 DMEM에 봉투 인코딩 플라스미드 NG 10 NG 문신 인코딩 (예 : PTAT-III 27)를 플라스미드와 150를 추가합니다. </li><li> 같은 pSVIII으로 문신에 의존 봉투를 코딩하는 플라스미드를 사용하는 경우 문신 인코딩 플라스미드에만 필요합니다. </li><li> 튜브 B : 1에서 450 ng의 PEI를 (추가5 ㎕의 DMEM에 μg / μL 용액). </li><li> 철저하게 10 초 동안 볼 텍싱에 의해 튜브 A. 믹스에 관 B의 내용을 추가하고 형질 믹스에서 객실 온도 (22 ° C에서)에서 10 분을 품어. </li><li> 96 - 웰 플레이트 잘 당 형질 믹스 10 μl를 추가합니다. 37 ° C, 5 % CO 2에서 48 시간 동안 배양한다. </li></ol> 3 일 4 - ELISA <ol><li> 봉투 / 항체 복합체의 가능한 엔도 사이토 시스를 최소화하기 위해 실온에서 모든 실험을 수행합니다. </li><li> 동시에 사용되는 접시 당 세척 완충액 250 ㎖를 준비한다. 세척 버퍼 1X 트리스 - 완충 생리 식염수 (TBS)의 pH 7.5 (50 mM 트리스 - 염소, pH가 7.5, 150 밀리미터의 NaCl) 1 mM의 MgCl2를 1.8 mM의 염화칼슘이 보충. </li><li> 1 %의 탈지 분유 및 세척 버퍼에 5 mM 트리스 산도 8.0을 추가하여 접시 당 버퍼를 차단 125 ML을 준비합니다. </li><li> 96 웰 플레이트에서 세포 배양 매체와 형질 믹스 (뜨는)를 제거합니다. </li><li> 100 μ 추가; 잘 당 버퍼를 차단하고 실온에서 20 분을 품어의 리터. </li><li> 상층 액을 제거하고 버퍼를 차단에 적절한 농도로 희석 아니라 당 항체 (혈청)의 50 μl를 추가합니다. 통상적으로, 1 μg / ml의 농도를 사용한다. 실온에서 1 시간을 품어. </li><li> 100 μL가 버퍼를 차단 한 후 100 ㎕의 세척 버퍼로 세척 공정의 3 배를 반복 씻을 배. </li><li> , 상층 액을 제거 버퍼를 차단 100 μl를 추가하고, 실온에서 5 분을 품어. </li><li> 상층 액을 제거하고 버퍼를 차단에서 1 / 3000로 희석 차 항체의 50 μl를 추가합니다. 제조 업체의 차이에 따라 최적의 항체 희석 다릅니다. 실온에서 40 분을 품어. </li><li> 100 μL가 버퍼를 차단 한 후 100 ㎕의 세척 버퍼로 세척 공정의 3 배를 반복 씻을 배. </li></ol> (4) 데이터 수집 <ol><li> 판으로부터 상층 액을 제거하고 잘 당 30 ㎕의 1 배 강화 된 화학 발광 (ECL) 기판을 추가합니다. </li><li> 화학 획득1 초 동안 iluminescence 신호 / 웰에 대한 제조사의 지시에 따라 적절한 플레이트 리더. 시간을 읽는 것은 하드웨어의 차이에 따라 다를 수 있습니다. </li></ol>

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Suppl. 16E (Q449), (1992).</li><li>Bonsignori, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-Dependent+Cellular+Cytotoxicity-Mediating+Antibodies+from+an+HIV-1+Vaccine+Efficacy+Trial+Target+Multiple+Epitopes+and+Preferentially+Use+the+VH1+Gene+Family.">Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family.</a> J. Virol. 86, 11521-11532 (2012).</li><li>Brand, D., Srinivasan, K., Sodroski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+human+immunodeficiency+virus+type+1+entry+in+the+CDR2+loop+of+the+CD4+glycoprotein.">Determinants of human immunodeficiency virus type 1 entry in the CDR2 loop of the CD4 glycoprotein.</a> J. Virol. 69, 166-171 (1995).</li></ol>

저자는 관심 없음 충돌 선언하지 않습니다.

이 분석은 HIV-1 삼량 봉투는 세포 표면에 발현하여 특정 모노클로 날 항체의 상호 작용을 검출하기 위해 최적화된다. 프로토콜이 확립 한 후에는 전체적으로 낮은 재료 비용 및 항체의 적은 양의 높은 처리량으로 매체에서 사용될 수있다. 이 분석은 형질 기반이기 때문에, 용이하게 봉투의 형태에 미치는 영향을 연구하기 위해 같은 CD4와 같은 세포 단백질의 동시 발현을 위해 적응 될 수있다. <p...

삼량 바이러스 엔벨로프 당 단백질 (봉투)에 의해 매개되는 인간 면역 결핍 바이러스 1 형 (HIV-1) 항목은 전염성주기의 첫 번째 단계입니다. 비리의 표면에서 발표 만 노출 바이러스 항원이기 때문에, 봉투 삼량은 중화 및 nonneutralizing 항체를 이끌어 낸다. 따라서, 그것은 백신의 면역원 디자인에 대한 흥미있는 후보를 나타냅니다. 그러나, 수용성 또는 재조합 형태로 봉투와 함께 예방 접종 시험 차 HIV-1가 1-3 분리 대부분에 대한 최소한의 효과와 반응을 이끌어 냈다. 그럼에도 불구하고, 부분적인 효능 면역원 후보로 HIV-1 봉투의 RV144 백신 재판 4 새로운 관심에서 관찰. 이는 백신 유도 항 봉투 항체는 SIV에 대한 보호의 어느 정도를 생성하기에 충분했고, HIV 5 도전 것을 나타내는 최근 연구에 의해 확증되었다. 소포체에서 합성되고, 봉투의 glycoprote 후전구체, gp160에, 바이러스 성 융합 프로세스 연료하는 능력에 중요한 다양한 번역 후 변형을 겪는다. 봉투 전구체는 gp120의-gp41의 연락을 유지 비공유 상호 작용으로, 그 여분의 세포질 gp120의 및 횡단 gp41 서브 유닛 6-10로 분해되기 전에 삼량에 적절하고 동료 접어해야합니다. 감염된 세포는 주로 기계의 전체 질량 (11, 12)의 50 % 정도를 포함하는, 봉투 glycosylating을 담당한다. 더 나은 다른 입체 형태를 이해하는 중요성을 강조 봉투는 적응하고, 그렇지 않으면 15 ~ 19 노출 될 수있는 특정 높은 면역 원성 항원을 숨길 수 있도록 생각하는 준 안정성을 제공하면서 그 결과 복잡한 구조는, 봉투는 13, 14 구조적으로 유연하게 할 수 있습니다 기본 봉투 삼량 체에 의해 샘플링. 지금까지 여러 가지 기술들이 개발되고 성공적 봉투 구조적 CH를 연구하는데 사용되어왔다Anges의. 그러나, 그들은 종종 특정 봉투 컨텍스트로 제한되고, 자신의 한계에 따라 다릅니다. 예를 들어, 표면 플라즈몬 공명 형태 또는 특정 모노클로 날 항체를 사용하여 면역 침전 분석법 (모노클로 날 항체)은, 하나는 자신의 삼량 체 형태 20,21 immunogenetically에서 상이한 것으로 알려진 모노머 용해 또는 가용화 된 봉투 분자에 의존한다. 최근의 연구는 분열은 주로 항체 14,22,23를 nonneutralizing 인식 항원의 노출이 봉투의 입체 형태에 영향을 미치는 것이 좋습니다. 여기에서 우리는 세부 사항에서 cellularly 발현 봉투의 삼량 18,24-26의 형태를 빠르고 쉽게 결정을 허용하는 방법을 설명합니다. 인간 점착성 세포주에서 봉투의 일시적인 형질 감염에 이어 봉투 특정 항체의 결합은 단순한 화학 발광 반응을 이용하여 검출된다. 이 기술은 또한 환경의 구조적 특성을 사용할 수있는 형태를-의존적이다덴트 항체. 따라서, 이러한 분석은 강력하고 매우 유연한 검출 방법을 제공한다.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HOS cells</td>
			<td style="width:103px;">ATCC</td>
			<td style="width:119px;">CRL-1543</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">White Opaque Tissue Culture Plate, 96 well, Flat Bottom</td>
			<td style="width:103px;">BD</td>
			<td align="right" style="width:119px;">353296</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polyethylenimine, linear, 25000 MW</td>
			<td style="width:103px;">Polysciences</td>
			<td align="right" style="width:119px;">23966</td>
			<td>Prepared in 1mg/ml solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:103px;">Invitrogen</td>
			<td align="right" style="width:119px;">11995</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Goat Anti-Human IgG, Peroxidase Conjugated</td>
			<td style="width:103px;">Pierce</td>
			<td align="right" style="width:119px;">31413</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Enhanced Chemiluminescence Substrate</td>
			<td style="width:103px;">PerkinElmer</td>
			<td style="width:119px;">NEL105001EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TriStar LB 941, Plate Reader</td>
			<td style="width:103px;">Berthold Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

상술 한 일반적인 절차를 사용하여, 우리는 야생형 (중량) 또는 돌연변이 된 봉투 하나에 CD4i 에피토프의 노출에 가용성 CD4 (sCD4)과 동시 발현 세포 CD4의 영향을 검정 프로토콜을 적응 18,24 전술 한 바와 같이, 25, 28. 한 개략적 일반적 절차와 sCD4 또는 CD4 세포 (18)의 동시 발현에 의해 치료 다음 CD4i 에피토프의 노출을 나타내는 도표. 18,24 예상대?...

Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

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 ...

conformational-evaluation-hiv-1-trimeric-envelope-glycoproteins-using

Research

JoVE Journal

Immunology and Infection

14.2K 조회수.  Université de Montréal. 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.

비디오: Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

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 ...

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Cryo - 전자 Tomography 및 자동 하위 tomogram 평균을 사용하여 HIV의 봉투 Glycoproteins의 분자 구조 결정

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

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 ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

의 이미징 HIV-1 봉투 유발 Virological 시냅스와 합성 지질 Bilayers에 신호

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

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 ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomo을 사용하여 전자 Cryotomography 재건에서 바이러스 성 봉투 ​​당 단백질 스파이크의 평균화

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

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. ...