Name: evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy
Uploaded: 2016-09-05T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

O trabalho aqui apresentado foi apoiado pelos Institutos Canadenses de Pesquisa em Saúde (CIHR) (concede DCB-120.266, PPP-133377 e HBF-348967 para AG).

1. Células e transfecções <ol><li> Culturas de células HEK 293T em meio de Eagle modificado por Dulbecco (DMEM) suplementado com 10% de soro fetal de bovino (FBS) e 1% de penicilina / estreptomicina. Prepara-se uma suspensão de 2 x 10 5 células / mL no meio de cultura celular. Adicionar 500, 100 e 1.000 mL da suspensão de células a cada poço de 24 poços, 96 poços e 12 poços placas, para a produção virai, a viabilidade celular e ensaios de activação imunitária, respectivamente (Figur...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+host+ICAM-1+in+laboratory+and+clinical+strains+of+human+immunodeficiency+virus+type+1+increases+virus+infectivity+and+CD4(+)-T-cell+depletion+in+human+lymphoid+tissue,+a+major+site+of+replication+in+vivo.">Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.</a> J Virol. 76, 1004-1014 (2002).</li><li>Shan, L., 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=A+novel+PCR+assay+for+quantification+of+HIV-1+RNA.">A novel PCR assay for quantification of HIV-1 RNA.</a> J Virol. 87, 6521-6525 (2013).</li><li>Clerzius, G., 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=The+PKR+activator,+PACT,+becomes+a+PKR+inhibitor+during+HIV-1+replication.">The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication.</a> Retrovirology. 10, 96 (2013).</li><li>Bradford, M. 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O ensaio de produção de HIV-1 descrita foi realizada utilizando células HEK293T (Figura 2) e é semelhante para os ensaios utilizados para rastrear ARN de HIV-1 para ribozima eficaz 13, shRNA 10,29, ARNsi 30, e U1i locais de ARN alvo 11,31. Usando diferentes métodos para quantificar a produção de HIV-1, a maioria dos estudos mediram a produção virai de 48 h após a co-transfecção de um plasmídeo de expressão do VIH-1 com RNAs candidatos. Após a pr...

Uma limitação dos actuais tratamentos do HIV-1 é que eles devem ser cronicamente administrado para prevenir a progressão da doença. Transplantação de VIH-1 resistente a linfócitos T, ou células-tronco hematopoiéticas, tem o potencial para proporcionar um controlo de longa duração de replicação do HIV-1 na ausência do tratamento medicamentoso 1,2 e pode também ser uma abordagem eficaz para atingir uma cura de HIV-1 3. Uma maneira de tornar as células resistentes ao HIV-1 é a replicação para inserir um ou mais genes que codificam para anti-HIV-1 RNA ou péptidos em células de um indivíduo infectado durante um transplante autólogo 4. Vários genes candidatos anti-VIH-1 foram concebidos com alguns estudos clínicos que entram em combinações de dois ou três 5 6, para prevenir o desenvolvimento de resistência do HIV-1 para um único gene. Anti-HIV-1 RNA estão entre os principais candidatos para a terapia de combinação de genes devido ao seu baixo potencial para provocar respostas imunes e porquesão transcritas a partir de sequências de genes muito curtos. Alguns-HIV-1 anti RNAs foram projetados para direcionar entrada e de integração viral. No entanto, a maioria dos ARNs anti-HIV-1 como alvo passos pós-integração no ciclo de vida viral (Figura 1). Inibidores pós-integração incluem RNAs de engodo, tendo como alvo os HIV-1 proteínas reguladoras Tat ou Rev 1, e RNAs à base de anti-senso, tendo como alvo locais diferentes em HIV-1 RNA, como as ribozimas 7, 8 e shRNAs U1i RNAs 9. Os métodos que foram utilizados para comparar a eficácia dos anti-HIV-1-RNA incluem a monitorização de replicação virai nas células transduzidas com genes que codificam para os ARN candidatas e medindo a produção virai em células transitoriamente transfectadas com plasmídeos que expressam os ARN candidatas e um plasmídeo de expressão de HIV-1 10 -13. Nós já usou um ensaio de produção HIV-1 para a tela HIV-1 RNA para sites de destino nova ribozimas 13-15. Estes métodos já foram refinados para optimizar o formato de um ARNmolécula de interferência expressa a partir de DNA de plasmídeo como um shRNA ou entregue como um siARN sintética 16. O ensaio mede a produção de vírus maduras a partir de células 293T de rim embrionário humano (HEK), e pode ser usado para comparar os efeitos dos inibidores que têm como alvo as etapas de pós-integração no ciclo de replicação do HIV-1 (Figura 1). Para os inibidores que têm como alvo as etapas de pré-integração, ensaios alternativos, tais como um ensaio de infecciosidade de células TZM BL-17 são necessários para avaliar a eficácia antiviral. As principais preocupações de segurança para a entrega de anti-HIV-1 RNA na clínica incluem potenciais efeitos fora do alvo em RNAs humanos ou proteínas, e ativação de sensores imune inata. Para avaliar a toxicidade do anti-HIV-1 siRNAs, utilizou-se um ensaio de viabilidade celular em diferentes linhas celulares 16. Nós também medido activação dos sensores imunes ARN de cadeia dupla, ARN activada proteína quinase R (PKR) e Toll like receptor 3 (TLR3), bem como expressão do gene de interferão estimulada, p150 ADAR1. Estes ensaios podem ser utilizados para confirmar que a eficácia do anti-HIV-1 RNA não é devido aos efeitos indirectos sobre a viabilidade celular ou a activação do sensor imune. Eles são também úteis na exclusão com RNAs candidatos potenciais toxicidades de desenvolvimento posterior. Nos seguintes protocolos, os procedimentos para identificar novos RNAs terapêuticos e optimizar o formato das existentes são descritos. Os métodos são úteis para inibidores pós-integração baseadas em ARN rastreio de HIV-1 da replicação e pode ser adaptado para o rastreio de outros inibidores pós-integração, tais como pequenas moléculas de direccionamento Rev exportação mediada por ARN viral 18 ou CRISPR / sistemas Cas concebidos para direccionar integrada HIV-1 DNA 19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin Gibco</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates, 96 well, 24 well, 6 well.</td>
			<td>Corning</td>
			<td>353075, 353047, 353043</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tubes Axygen</td>
			<td>Corning</td>
			<td>311-08-051</td>
			<td></td>
		</tr>
		<tr>
			<td>Low molecular weight Poly I:C</td>
			<td>InvivoGen</td>
			<td>3182-29-6</td>
			<td></td>
		</tr>
		<tr>
			<td>DharmaFECT-1</td>
			<td>Dharmacon</td>
			<td>T-2001-01</td>
			<td>transfection reagent for synthetic RNAs</td>
		</tr>
		<tr>
			<td>TransIT-LT1</td>
			<td>Mirus</td>
			<td>MIR 2300</td>
			<td>transfection reagent for RNA expression plasmids</td>
		</tr>
		<tr>
			<td>Nonidet P40 (NP-40)</td>
			<td>USB</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr>
			<td>[32P]dTTP</td>
			<td>Perkin Elmer</td>
			<td>BLU505H</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(A) RNA template&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10108626001</td>
			<td></td>
		</tr>
		<tr>
			<td>oligo(dT)12-18 DNA primer</td>
			<td>Thermo Fisher</td>
			<td>18418-012</td>
			<td></td>
		</tr>
		<tr>
			<td>DEAE filtermat paper&#160;</td>
			<td>Perkin Elmer</td>
			<td>1450-522</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate scintillation counter</td>
			<td>Perkin Elmer</td>
			<td>1450-024</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Sigma-Aldrich</td>
			<td>M-2128</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate spectrophotometer</td>
			<td>Bio-rad</td>
			<td>1706930</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis buffer tablets</td>
			<td>Roche</td>
			<td>4693159001, 4906837001</td>
			<td>protease and phosphatase inhibitors</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Bio-rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel running chamber</td>
			<td>Hoefer</td>
			<td>SE600</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-dry transfer cell</td>
			<td>Bio-rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein ladder EZ-Run</td>
			<td>Thermo Fisher</td>
			<td>BP3603-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Bio-rad</td>
			<td>162-0094</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody stripping solution</td>
			<td>Millipore</td>
			<td>2504</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL - Pierce</td>
			<td>Thermo Fisher&#160;</td>
			<td>PI32106</td>
			<td></td>
		</tr>
		<tr>
			<td>ADAR1 antibody</td>
			<td></td>
			<td></td>
			<td>from Dr. B.L. Bass</td>
		</tr>
		<tr>
			<td>phospho-T446-PKR antibody</td>
			<td>Abcam</td>
			<td>ab32036</td>
			<td></td>
		</tr>
		<tr>
			<td>phospho-S396-IRF3 antibody</td>
			<td>Cell Signaling</td>
			<td>4947</td>
			<td></td>
		</tr>
		<tr>
			<td>PKR antibody</td>
			<td></td>
			<td></td>
			<td>from Dr. A. Hovanessian</td>
		</tr>
		<tr>
			<td>IRF3 antibody</td>
			<td>Cell Signaling</td>
			<td>11904</td>
			<td></td>
		</tr>
		<tr>
			<td>Actin antibody</td>
			<td>Millipore</td>
			<td>MAB1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-labeled goat anti-rabbit</td>
			<td>KPL</td>
			<td>474-1506</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-labeled goat anti-mouse</td>
			<td>KPL</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>6226-79-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Um esquema geral dos processos é mostrado na Figura 2, com um plano de transfecção exemplo para três RNAs de teste e um ARN de controlo fornecida na Figura 2B. Para os ensaios de viabilidade celular de produção e virais, a leitura para cada construto de teste é normalizada para um controlo negativo. Os duplicados estão transfectadas em conjuntos, de modo que cada ARN teste é normalizado para o seu controlo negativo adjacente. Isto é feito para ...

Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

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

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.

Avaliação da eficácia e toxicidade de RNAs Segmentação HIV-1 Produção para uso em Gene ou Quimioterapia

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 ...

The overall goal of this procedure is to compare the efficacy and toxicity of new RNAs targeting HIV-1 production. This method can help identify new RNA molecules for using HIV-1 gene or drug therapy, such as short hairpin or small interfering RNAs, ribozymes, and RNA decoys. The main advantage of this technique is that several new RNA designs can be screened simultaneously with a quick turnaround time.Although this method was designed to evaluate new RNA drug or gene therapy for HIV, it can also be applied to other systems, such as small molecules or CRISPR tasks. We first had the idea for this method when we were setting up experiments to screen a large number of ribozymes for anti-HIV effects. To begin, prepare a suspension of human embryonic kidney 293T cells at two times 10 to the fifth cells per milliliter in DMEM with 10%FPS and 1%penicillin streptomycin.Add 500, 100 and 1, 000 microliters of the cell suspension to each of the wells of 24, 96, and 12-well plates for viral production, cell viability, and immune activation assays, respectively. Gently swirl the plates and incubate them overnight at 37 degrees Celsius with 5%carbon dioxide to 50%to 70%confluency. For the viral production assay, prepare a 10 nanogram per microliter dilution of an HIV-1 expression plasmid and add 10 microliters to each assay tube.Next, prepare five micromolar dilutions of test RNAs and a negative control RNA. Then add 2.5 microliters or 10 microliters of each test RNA and negative control dilution to the corresponding tubes for 25 nanomolar or 100 nanomolar final concentrations. For the cell viability assay, prepare five micromolar dilutions of test RNAs and a 10 milligram per milliliter dilution of the positive control RNA, low molecular weight poly(I:C)Add two microliters of the test RNA or the positive control RNA dilutions to the corresponding tubes for 100 nanomolar or 200 micrograms per milliliter final concentrations, respectively.For the immune activation assay, add 20 microliters of test RNA or positive control RNA dilutions, as prepared for the cell viability assay, to the corresponding tubes for 100 nanomolar or 200 micrograms per milliliter final concentrations, respectively. Then add 50, 25, or 75 microliters of DMEM to each transfection tube for viral production, cell viability, and immune activation assays, respectively. For viral production assays, bring cells and prepared transfection tubes to a biosafety level three, or BSL-3, laboratory before the next step.Add two microliters of the transfection reagent sequentially to the transfection tubes, and incubate 15 to 20 minutes to allow complexes to form. Add the entire transfection mixture from each microtube drop-wise to the corresponding positions in the cell culture plates. Gently swirl, and incubate the plates for 48 hours at 37 degrees Celsius and 5%carbon dioxide.To carry out the viral production assay, after removing the 24-well cell culture plates from the incubator, transfer them to the BSL-3 cell culture hood. Gently swirl the plates and then transfer 150 microliters of the supernatant from each well to a corresponding well in a 96-well flat-bottom plate to be used to quantify HIV-1 production. Transfer five microliters of supernatant to the corresponding wells in a 96-well plate containing 25 microliters of a viral disruption cocktail.After incubating the mixture for five minutes at room temperature, transfer the plate to a radioactivity work station. Next, prepare a radioactive cocktail, and add 25 microliters to each well of viral supernatant and disruption cocktail. Incubate the plates at 37 degrees Celsius for two hours.Spot five microliters of the reaction mixture onto corresponding squares in a glass fiber/DEAE filter mat paper, and allow the spots to dry for 10 minutes. Use 2x SSC buffer to wash the papers five times for five minutes each, followed by two one-minute washes with 95%ethanol. Allow the papers to dry before sealing them in sample bags.Clip sample bags containing the filter mat paper into a cassette, and insert the cassette into a microplate scintillation counter. Then start the counter. For any viral quantification method, divide the values obtained for each test RNA by the adjacent negative control and multiply this value by 100 to get the percent inhibition of HIV-1 production for each replicate of the test RNAs.To carry out a cell viability assay, after removing the 96-well plates from the incubator, add 20 microliters of five milligrams per milliliter MTT and DPBS to each well. Then incubate the plates for three hours at 37 degrees Celsius. Add 150 microliters of acidified isopropanol with detergent to each well, and incubate the plates at room temperature for two hours.Then use a microplate spectrophotometer to determine the absorbance at 570 nanometers. Calculate the relative MTT metabolism for each positive control and test RNA by dividing the value obtained for each sample by its adjacent transfection control. For the immune activation assay, after removing the 12-well plates from the incubator, aspirate the culture medium and use DPBS to gently wash the cells twice.Then add 70 microliters of cold lysis buffer containing protease and phosphotase inhibitors to each well, and incubate the plates on ice for 10 minutes. Transfer cell lysates to microtubes, and fast freeze them by immersing the tubes in liquid nitrogen. After allowing the samples to thaw, freeze-thaw them two more times, for a total of three freeze-thaw cycles.Next, centrifuge the lysates at 15, 700 times g and 4 degrees Celsius for 15 minutes to pellet the cell debris. Then, following electrophoresis and transfer to a membrane, reveal protein bands by incubating the membrane in Ponceau S for one minute. Then wash it with double-distilled water.Use the bands and protein ladder as a guide to cut the membrane at 80 and 55 kilodaltons. Now, use TBS with 0.05%TWEEN 20 or TBST to wash out the Ponceau S staining. Add TBST with 5%non-fat milk to completely cover the membranes, and incubate them at room temperature with agitation for one hour.Transfer the membranes into TBST with 3%BSA and antibodies diluted 1:1, 000 for ADAR, phosphor-PKR, and phosphor-IRF3 for the top, middle, and bottom pieces of the membrane, respectively. Incubate at 4 degrees Celsius with agitation overnight. The following morning, use TBST to wash the membranes five times for five minutes each, before transferring to TBST with 5%non-fat milk and 1:5, 000 peroxidase labeled goat anti-rabbit secondary antibodies for one hour.After visualizing the protein bands on films, use a stripping solution to wash the middle and bottom pieces of the membrane for 10 minutes, before incubating the middle piece of the membrane in PKR antibodies and the bottom piece in IRF3 antibodies. As a positive control, probe the bottom membrane with antibodies to Actin according to the text protocol. A general schematic of the assays is shown here with an example transfection plan for three test RNAs and a control RNA.For viral production and cell viability assays, the readout for each test construct is normalized to a negative control. Using the HIV-1 production assay in cells transfected with a plasmid expressing HIV-1 strain NL4-3, the percent of RT activity in cells co-transfected with one of the siRNAs shown here, compared to cells transfected with a long Dicer substrate nonsense siRNA, was calculated to identify the optimal length of RNA interference molecules targeting a conserved site in HIV-1 RNA. Using the most effective siRNAs identified, the percent metabolism of MTT was determined for transfected cells.Along double-stranded RNA, poly(I:C)reduced cell viability only at the highest dose. No significant reduction in cell viability was observed for siRNAs targeting the 1498 site in HIV-1 RNA, regardless of their length. As demonstrated here by Western blot, the same set of RNAs was evaluated in the immune activation assay.Under conditions where poly(I:C)activated the expression of ADAR1-p150 and induced phosphorilation of PKR and IRF3, no significant effect on any inactivation markers could be observed for any of the test RNAs. Once mastered, this technique can be done in seven days, if it's performed properly. While attempting this procedure, it is important to use high quality preparations of both RNA and DNA for transfection.Furthermore, to include the proper negative and positive controls. Following this procedure, other methods like HIV infection of transduced cells, can be used to evaluate the long-term efficacy and toxicity of potential anti-HIV candidates. After its development, this technique allowed us to identify an accessible and conserved target site in HIV RNA and optimize the formats of ribozymes, shRNAs and siRNAs targeting it.After watching this video, you should have a good understanding of how to quickly screen new RNA candidates targeting HIV production for efficacy using the viral production assay, and potential toxicity using the immune activation and cell viability assays. Don't forget that working with HIV and radiolabeled nucleotides can be extremely hazardous. Precautions such as using certified biosafety and radioactivity laboratories should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Inferência genotípica do HIV-1 Tropismo Usando de base populacional de Seqüenciamento 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 ...

Imunologia e Infecção

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Geração de células T específicas Multivirus para Prevenir / tratar infecções virais após transplante de células-tronco hematopoéticas alogênico

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

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

Amplificar e Quantificação do HIV-1 RNA no indivíduos infectados pelo HIV com carga viral abaixo do limite de detecção pela Standard Ensaios Clínicos

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 ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

Em desencapsulamento vitro do 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 ...

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

Imagem do HIV-1 Synapse Envelope induzida virológica e Sinalização em bicamadas lipídicas sintéticas

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

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

Avaliação conformacional do HIV-1 trimérico glicoproteínas do envelope Usando um ELISA Ensaio Celular

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

De alto rendimento quantitativo em tempo real de RT-PCR Ensaio para Determinar Perfis de Expressão do tipo I e III do interferão subtipos

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Isolamento de exossomas a partir do plasma de indivíduos HIV-1 positivos

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. ...

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

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

Medição de<em&gt; In Vitro</em&gt; Actividade integração do HIV-1 de pré-integração Complexos

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 ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Sistema de Eficácia e citotoxicidade Rastreio de inibidores Segmentação intracelular<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...