We thank Dr Jamil Jubrail for reading the manuscript. This work was supported by grants from CNRS, Inserm, Universit&#233; Paris Descartes, Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM DEQ20130326518 including a doctoral fellowship for GLB) and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales (ANRS, including a post-doctoral fellowship for CD) to FN. A. Dumas was supported by doctoral fellowships from Universit&#233; Paris Descartes and Sidaction.

O protocolo tem de ser efectuada em estrita conformidade com as legislações nacionais e internacionais e regulamentos locais. O sangue de doadores saudáveis ​​que deram o seu consentimento para doar sangue para fins de pesquisa foi obtido a partir de centros de transfusão de sangue com o qual as instituições assinaram acordo. protecções especiais devem ser tomados ao usar sangue humano. As experiências com o HIV-1 deve ser realizado em um nível de segurança biológica, ou 2 3 (BSL-3 ou 2) laboratório de acordo com a legislação local. 1. Preparação de macrófagos humanos Monócitos-derivados (hMDMs) por centrifugação em gradiente de densidade e por adesão Selecção <ol><li> Comece com o sangue fresco de dadores saudáveis ​​(9 mL). Dilui-se todo o volume de sangue fresco com 1x estéril salina tamponada com fosfato (PBS) sem Ca 2+ e Mg 2+ para se obter um volume final de 70 ml e adicionar cuidadosamente o sangue diluído em dois tubos de 50 mL cónicos (35 mL por tubo) , no topo de 15 ml de umaeutral, altamente ramificado, de elevada massa, polissacárido hidrófilo na solução já em cada tubo. </li><li> Centrífuga ambos os tubos de sangue a 537 xg durante 20 min a 20 ° C sem travão. Em seguida, recolher as células mononucleares do sangue periférico (PBMCs) contidos no anel célula nebuloso na interface e transferi-las para um novo tubo de 50 ml contendo 15 ml de PBS 1x sem Ca 2+ e Mg 2+. </li><li> Centrifugar as células a 218 xg durante 5 min a 20 ° C e ressuspender o sedimento em 45 ml de PBS 1x sem Ca 2+ e Mg 2+. </li><li> Centrifugar as células a 218 xg durante 5 min a 20 ° C, ressuspender o sedimento em 10 ml de PBS 1x sem Ca 2+ e Mg 2+, e contar as células por diluição para uma diluição final de 1/200 em azul de tripano. </li><li> Centrifugar as células a 218 xg durante 5 min a 20 ° C e ressuspender o sedimento em meio RPMI (Roswell Park Memorial Institute) 1640 suplementado com 2 mM de L-glutamina e 100 ug / ml Penicillin-estreptomicina a ter 7 x 10 6 PBMC por cavidade em 2 ml de meio em cada poço de uma placa de 6 bem. </li><li> Incubar as placas a 37 ° C com 5% de CO 2 durante 2 h. Após 2 h os monócitos vai aderiram ao plástico. </li><li> Para permitir que os recém-isolados monócitos de se diferenciar em hMDMs, Aspirar o meio e substitui-la com 2 ml hMDM meio (RPMI 1640, 10% descomplementado Soro Fetal de Vitela (FCS), 2 mM de L-glutamina e 1% de penicilina-estreptomicina) suplementado com factor de estimulação do macrófago humano recombinante Colony (RHM-CSF), a uma concentração final de 10 ng / ml. </li><li> Incubar as células a 37 ° C com 5% de CO 2 durante 11 dias (Figura 1). </li><li> No dia 11, remover o meio e lava-se 2 vezes com 2 ml de meio por poço hMDM frio. De seguida, lavar cada poço 2 vezes com 1 ml de PBS frio 1x. </li><li> Para retirar hMDMs totalmente diferenciadas, lavar cada poço 1 hora com 1 ml de PBS frio 1x com ácido ethylenediaminetetraacetatic 2 mM (EDTA) e incuba-se as células em 2 ml de PBS frio 1x com EDTA 2 mM por poço, durante 15-60 min a 4 ° C. NOTA: Os métodos alternativos para separar existem as células (por exemplo, tripsina ou atividades de tripsina-like) que possa dar bons resultados 11. </li><li> Depois de descolamento (concluído com cuidado pipetando cima e para baixo no poço), recolher as células e colocá-los em um tubo de 50 ml de gelo contendo 10 ml de meio hMDM frio. </li><li> Centrifugar as células a 218 xg durante 5 min a 20 ° C, ressuspender o sedimento em 10 ml de meio frio hMDM, e contar as células por diluição de 1/20 em azul de tripano. </li><li> Semear as células a 1 x 10 6 hMDMs por 35 milímetros de vidro da classe microscopia prato inferior e incubar as placas a 37 ° C com 5% de CO 2 durante 1 dia. </li></ol> 2. Produção e quantificação do VIH-1 reservas virais NOTA: NLR4.3 HIV-1 Gag-IGFP (Green Fluorescent Protein) carregando um envelope R5-trópico, presente de M. Schindler 10 é utilizado para infectar macrófagos e células infectadas para ver em tempo real. <ol><li> Produção de reservas virais através de transfecção de células humanas embrionárias de rim 293T (2 x 10 6 em 100 milímetros prato) com 6 ug do ADN pró-viral correspondente usando um reagente de transfecção comercial. </li><li> Quantificar a infecciosidade das reservas de vírus com as células indicadoras HeLa TZM-bl (possuindo o gene ß-galactosidase sob o controlo do HIV-1 LTR) utilizando diluições em série dos stocks virais, seguido de uma coloração ß-galactosidase das células e contagem células de azul 12. </li></ol> 3. A infecção de hMDMs com HIV-1 <ol><li> Adicionar o vírus a uma multiplicidade de infecção (MOI) 0,02-0,03 em 1 ml de meio para hMDM hMDMs (células plaqueadas em 1,13). Para controlar poços adicionar apenas 1 ml de meio hMDM e incubar os pratos a 37 ° C com 5% de CO 2 durante 2 dias (Figura 1). </li><li> No dia 2 lavar as células com hMDM medio 3 vezes e adicionar 1 ml de meio hMDM fresco por prato. Incubar as células a 37 ° C com 5% de CO 2 durante 6 dias (Figura 1). </li></ol> 4. Opsonização de ovelhas Glóbulos vermelhos <ol><li> Para a preparação de 7 x 10 6 por placa de SRBCs, lavar os SRBCs duas vezes em 100 ul de solução contendo 0,1% de Albumina de Soro Bovino (BSA) em PBS 1x com centrifugação a 600 x g durante 4 min. </li><li> Ressuspender as SRBCs lavadas com 500 ul de 1x PBS / BSA a 0,1% com IgG de coelho anti-SRBCs a uma concentração sub-aglutinação por 5 ul de SRBCs e incubar com rotação à temperatura ambiente durante 30 min. NOTA: Para determinar a concentração sub-aglutinação de anti-IgG de SRBCs, preparar diluições em série de IgG (concentração de estoque de 13,1 mg / ml) a partir de 1/50 a 1 / 25.600 em 20 ul numa placa de 96 poços. Adicionar 2 x 10 6 SRBCs em 20 ul em cada cavidade e colocar a placa de um quarto escuro durante várias horas. A concentração sub-aglutinação é adiluição do poço imediatamente antes do poço com aglutinação (IgG + SRBCs formando uma rede). </li><li> Após rotação, centrifugar os SRBCs-IgG-opsonizadas a 600 xg durante 4 min e lava-se com 100 ul de 1x PBS / BSA a 0,1%, com centrifugação a 600 x g durante 4 min. </li><li> Ressuspender as SRBCs-IgG-opsonizados em meio pré-aquecido Fenol RPMI sem vermelho suplementado com 2 mM de L-glutamina e 1% de penicilina-estreptomicina (1 ml / prato). </li></ol> 5. células vivas Vídeo Microscopy fagocitose Assay <ol><li> Usar um sistema de imagiologia confocal, tais como um sistema de disco giratório equipado com uma câmara de aquecimento a 37 ° C com CO 2 passando através de um frasco com água para a humidificação. </li><li> Ligar a câmara de aquecimento antes da experiência ter a platina do microscópio a 37 ° C antes do início do ensaio de fagocitose. Ligue o microscópio e computador e carregar o software de imagem. </li><li> Otimizar as configurações de imagem, como velocidade de digitalização, Magnificatião, resolução, etc, para ter pelo menos uma célula por campo e a imagem de um quadro de cada minuto entre 60 a 120 min. NOTA: Aqui, a amostra é fotografada um quadro a cada minuto durante 60 min com 63x lente. </li><li> Coloque o prato de imagem no palco microscópio. Ajustar o foco e o local para encontrar apenas um conjunto de macrófagos infectados pelo HIV-1 no campo. Use as configurações de excitação / emissão adequadas com base no sistema de imagem usados ​​e sonda. Incluir um canal brilhante campo (BF) para observar phagosomes (Figura 1ii). Optimizar a aparência das diferentes imagens de canal, ajustando a percentagem de tempo de exposição a luz e transmitidos. NOTA: Aqui, NLR4.3 HIV-1 Gag-IGFP estava animado usando um laser de 491 nm com 50 ms de tempo de exposição com 20% do laser (Figura 1-I). </li><li> Remover o prato de imagem e adicionar 1 ml de uma suspensão SRBC a 7 x 10 6 / ml SRBCs ao prato (Figura 1). </li><li> Centrifugar a 500 xg durante2 min à temperatura ambiente para sincronizar a fagocitose, registar o tempo no final desta centrifugação e devolver o prato para a fase. </li><li> Optimizar a captura de foco e GFP e imagens BF em z-pilhas (em toda a espessura da célula com um passo a distância de 0,3 um - 20 geralmente planos) a cada minuto durante pelo menos 1 h. Salvar o vídeo de lapso de tempo no arquivo de formato nativo do sistema de imagem utilizado. NOTA: Aqui, os filmes de lapso de tempo foram salvos no formato nativo, arquivos * .stk. </li></ol> 6. Análise da Time-lapse Filmes <ol><li> Para edição de vídeo, clique no menu drop-down &quot;Apps&quot; e na guia &quot;Análise multidimensional de dados&quot; no software de edição de vídeo. <ol><li> Para abrir o arquivo, clique em &quot;Selecionar Arquivo Base&quot;, em seguida, em &quot;Selecionar Directory&quot;. No &quot;Conjuntos de dados&quot;, selecione a aquisição de analisar (em formato .nd) e clique em &quot;Ver&quot;. Os dados são representados numa tabela com o tempo em uma colunad Z-plano na linha. </li><li> Para representar a infecção em um Z-projeção (Figura 2, painéis da esquerda), selecione 491 nm de comprimento de onda na caixa &quot;comprimentos de onda&quot; e clique na aba &quot;Z projecção&quot; com &quot;todos os planos&quot;. </li><li> Para analisar uma seqüência de tempo, selecione o comprimento de onda BF na caixa &quot;comprimentos de onda&quot; e escolha o plano ideal no eixo Z para distinguir SRBCs externos (Figura 2, seta vermelha), SRBCs internos (Figura 2, círculo vermelho) e o núcleo ( Figura 2, círculo azul). </li><li> Para salvar as montagens de vídeo, clique em &quot;Selecção [do X]&quot; e depois em &quot;Load Image (s)&quot;. Por fim, salve as imagens carregadas em formato .tif e abri-los em seguida em software ImageJ. </li></ol></li><li> Use o plugin &quot;Rastreio Manual&quot; no software ImageJ para medir a posição do núcleo e os diferentes phagosomes observadas no canal BF (Figura 3). <ol><li> DowNload o plug-in &quot;Manual de Rastreamento&quot; no site do ImageJ. No ImageJ, abra o plugin e a sequência de imagens a serem analisadas (Figura 3, passo 1 e 2). </li><li> Introduzir as definições tais como &quot;intervalo de tempo&quot; que representa quantidade de tempo entre quadros adjacentes, eo &quot;calibração x / y&quot;, que representa a distância per pixel (Figura 3, passo 3). NOTA: Aqui, guardar a sequência de imagens com um intervalo de tempo de 1 minuto entre cada armação e a calibração x / y de 0,205 uM porque o zoom 63X e uma câmara com tamanho de pixel de 6,45 x 6,45? M 2 são usados. </li><li> Para iniciar o rastreamento, clique em &quot;Adicionar faixa&quot; (Figura 3, passo 4) e clique em um centro de SRBC no primeiro tempo quando é internalizado. O próximo quadro aparece automaticamente. </li><li> Continue a clicar sobre o centro SRBC em todos os quadros de ter posições diferentes durante o tempo (Figura 3, Passo 6 na caixa vermelha). <ol><li> Começar a controlar o núcleo que a sua posição em todos os quadros, clicando em seu centro. Próximo acompanhar os phagosomes (clicando em seu centro), um por um na época (frame) de sua interiorização, diferente em função do SRBCs. Para maior comodidade para ver SRBC no canal BF, use a janela &quot;Brilho e Contraste&quot; durante o acompanhamento (Figura 3, passo 5). </li></ol></li><li> Entre cada rastreamento SRBC, clique em &quot;Adicionar faixa&quot; (Figura 3, passo 4) para ter uma nova pista. O número de rastreamento será alterado na segunda coluna, &quot;Track n °&quot; da tabela de resultados (Figura 3, passo 6). </li><li> Salvar os dados em uma planilha para continuar para a próxima etapa da análise. </li></ol></li><li> Use um software de planilha para calcular a distância percorrida de phagosomes contendo SRBCs para o núcleo e a velocidade dos phagosomes durante os primeiros 5 minutos após a internalização de SRBCs (Fifigura 4). <ol><li> Abra a tabela de folha de cálculo e um novo arquivo de planilha. Transferir para este novo arquivo os seguintes parâmetros somente, tempo, o X e Y coordenadas do núcleo e todos os SRBCs (Figura 4A). </li><li> Para calcular a distância entre o núcleo e SRBCs com apenas as suas coordenadas, considerar o núcleo e uma SRBC num plano XY de coordenadas (Figura 4B). NOTA: A distância entre dois pontos é o comprimento do caminho de os ligar. No plano, a distância entre o núcleo e SRBC é dada pelo teorema de Pitágoras. <ol><li> Se C (a distância entre o núcleo e o SRBC) indica o comprimento da hipotenusa e a e b representam os comprimentos dos outros dois lados, expressar o teorema de Pitágoras como a equação de Pitágoras: <img alt="equação 1" src="/files/ftp_upload/54568/54568eq1.jpg" /> NOTA: Ao mesmo tempo, no ortonormal, a distância horizontalum é (x núcleo -x SRBC) e a distância vertical b é (Y-Y núcleo SRBC). </li><li> Assim, o cálculo da distância entre o núcleo e o SRBC (Figura 4A, a caixa roxo) por: <img alt="equação 2" src="/files/ftp_upload/54568/54568eq2.jpg" /> NOTA: multiplicar o valor de distância obtida (em pixels) por x / y de calibragem ter a distância em uM. Aqui, a calibração x / y é 0,205 uM. </li><li> Em cada tempo, subtrair a distância entre o núcleo e o SRBC à distância inicial (Figura 4C). </li><li> Traça-se a medição em função do tempo para os primeiros 5 min. Aplicar uma linha de tendência linear (Figura 5A) para a trama e determinar a inclinação da linha de tendência linear que representa a velocidade fagossoma para o núcleo durante os primeiros 5 minutos após a internalização SRBC. </li><li> Agrupar os dados para calcular o erro médio e de estatísticaos valores de velocidade e traçar-los em uma forma adequada, utilizando o software apropriado (Figura 5B). </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

Esta técnica tem várias etapas críticas. Em primeiro lugar, a preparação de hMDMs e a sua infecção por HIV-1 é crítica, porque a percentagem de infecção é dependente do dador. De nota, decidimos usar macrófagos que não são polarizados in vitro antes da infecção, pois o estado de macrófagos potencialmente encontradas pelo vírus in vivo não foi bem caracterizada medida. Nós verificamos a expressão de vários marcadores de superfície, o que indica que os macrófagos são nem nem M1 ...

Macrophages play a major role in the innate immune system and in homeostasis. They are professional phagocytes that internalize and eliminate pathogens and debris by a process called phagocytosis 1,2. The phagosome, the closed compartment that forms after the engulfment of particulate material, undergoes a series of fusion and fission events with endocytic compartments, leading to a degradative compartment called the phagolysosome. This compartment has an acidic pH, due to the acquisition of proton-pumping v-ATPases, contains hydrolytic enzymes and is enriched in lysosomal-associated membrane proteins (LAMPs). The maturation of phagosomes is accompanied by their migration on microtubules 3,4 towards the cell center to reach a perinuclear location where lysosomes are accumulated.
Many pathogens have been reported to hijack phagosome maturation, including bacteria with intracellular lifestyles that modify the vacuolar environment where they reside 5. The Human Immunodeficiency Virus (HIV)-1 targets macrophages in addition to T cells. As macrophages are resistant to the cytopathic effects of the virus, unlike T cells, they can be considered as a reservoir for the virus. In addition, macrophages infected with HIV-1 show defective phagocytic functions and contribute to the emergence of opportunistic diseases. In particular, severe invasive non-typhoidal Salmonella disease caused by Salmonella Typhimurium ST313 has been prevalent for the last three decades in sub-Saharan African children or adults infected with HIV 6. It has been estimated that the risk of developing tuberculosis is more than 20 times greater in people living with HIV than among those without HIV infection. 
For all these reasons, it is important to better define the molecular mechanisms underlying the phagocytic defects in HIV-infected macrophages. We have shown that the uptake of particulate material, opsonized particles, bacteria or fungi, was inhibited in HIV-infected macrophages 7. Given that this inhibition is partial, we then set out to analyze the fate of the internalized phagosomes in HIV infected human macrophages 8. Because phagosome maturation is tightly connected with migration to the cell center and fusion with lysosomes, a defect in phagosomal maturation can be due to modifications of the trafficking modalities in the infected cell. The method described here uses IgG-opsonized Sheep Red Blood Cells (IgG-SRBCs) as a model to target receptor-mediated phagocytosis and in particular receptors for the Fc portion of immunoglobulins (FcR). These particles are easier to image in bright field (BF) than latex beads because extracellular and intracellular SRBCs show different refraction properties 9. To measure the velocity of phagosomes moving towards the nucleus in HIV-infected macrophages, we used a fluorescent virus 10 and set up a simple manual tracking method that is described here. The method does not require advanced programming and simply uses ImageJ. It is amenable to adherent cells and any type of particle or pathogen that can be visualized with bright field microscopy or with fluorescent imaging.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Falcon 100mm TC-Treated Cell Culture Dish</td>
			<td style="width:124px;">Corning</td>
			<td style="width:111px;">353003</td>
			<td style="width:245px;">For viral production</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Glass Bottom Dishes 35 mm uncoated 1.5</td>
			<td style="width:124px;">MatTek corporation</td>
			<td style="width:111px;">P35G-1.5-14-C Case</td>
			<td style="width:245px;">For acquisition</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Falcon Tissue Culture Plates 6-well</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">Corning. Inc. 353934</td>
			<td style="width:245px;">For human monocyte-derived macrophages</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Ficoll-Plaque PLUS</td>
			<td style="width:124px;">Dominique Dutscher</td>
			<td style="width:111px;">17-1440-03</td>
			<td style="width:245px;">a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">DPBS, no calcium, no magnesium</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">14190-094</td>
			<td style="width:245px;">Room temperature</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) 1X, liquid (High Glucose)</td>
			<td style="width:124px;">GIBCO, Molecular probes</td>
			<td style="width:111px;">31966-021</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for HEK cells culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">61870-010</td>
			<td style="width:245px;">Conserved at 4&#176;C; for hMDMs culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">F&#339;tal Calf Serum (FCS)&#160;</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">CVFSVF0001</td>
			<td style="width:245px;">Conserved at -20&#176;C ; decomplemented</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">15140-122</td>
			<td style="width:245px;">Conserved at -20&#176;C ; for hMDMs culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">11835-105</td>
			<td style="width:245px;">Warm in 37&#176;C water bath before use ; for phagocytosis assay</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">FuGENE6 Transfection Reagent</td>
			<td style="width:124px;">Promega</td>
			<td style="width:111px;">E2692</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for viral production</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Sheep red blood cells (SRBCs)</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">DSGMTN00-0Q</td>
			<td style="width:245px;">Conserved in Alsever buffer at 4&#176;C before use</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Anti-sheep red blood cells IgG</td>
			<td style="width:124px;">MP Biomedicals</td>
			<td style="width:111px;">55806</td>
			<td style="width:245px;">Conserved at 4&#176;C</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Bovine Serum Albumin heat shock fraction, pH 7, &#8805;98%&#160;</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:111px;">A7906</td>
			<td style="width:245px;">Conserved at -20&#176;C</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Inverted microscope DMI600</td>
			<td style="width:124px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Confocal Spinning Disk Unit&#160; CSU-X1M1</td>
			<td style="width:124px;">Yokogawa</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">491 nm 50mW laser</td>
			<td style="width:124px;">COBOLT CALYPSO</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">HCX PL APO CS Objectif</td>
			<td style="width:124px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">Objective lens ; Magnification 100x ; Numerical aperture 1.40 ; Immersion oil</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">CoolSnap HQ2 (FireWire) Camera</td>
			<td style="width:124px;">Photometrics</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">Pixel size 6.45 x 6.45 &#181;m ; Definition 1392 x 1040 ; Encoding the image in 14 Bit</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Metamorph 7.7.5 software</td>
			<td style="width:124px;">Molecular Devices</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">For the control of the microscope</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">GraphPad Prism software</td>
			<td style="width:124px;"></td>
			<td style="width:111px;"></td>
			<td style="width:245px;">For the statistics analysis</td>
		</tr>
	</tbody>
</table>

phagosome migration and velocity measured in live primary human macrophages infected with hiv-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

Fagocitose mediada por FcR-hMDMs HIV-1 infectadas e não-infectadas é descrito aqui usando SRBCs IgG-opsonizadas como alvos modelo (Figura 1). Os passos críticos deste protocolo são a preparação de hMDMs ea infecção com HIV-1. Com efeito, o rendimento e a qualidade dos macrófagos diferenciados varia entre dadores, bem como a taxa de infecção com eficiências na gama de 10-40%. Além disso, a preparação de IgG-opsonizado-SRBCs também é importante para evitar...

Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Migração fagossomo e velocidade medida no Live primárias macrófagos humanos infectados com o HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

phagosome-migration-velocity-measured-live-primary-human-macrophages

Research

JoVE Journal

Immunology and Infection

8.1K visualizações.  Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité. We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes. 

Vídeo: Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

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.

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparação e Uso de HIV-1 infectados CD4 + T-Primária células como as células-alvo em Ensaios Natural Killer Celular citotóxicos

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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.

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

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.

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

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.

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 ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Um<em&gt; In vitro</em&gt; Modelo de Co-infecção para Estudar<em&gt; Plasmodium falciparum</em&gt;-HIV-1 Interacções em monócitos humanos primários derivados de células imunes

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

Novas ferramentas para expandir as células T reguladoras de indivíduos HIV-1-infected

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Estudo da fagolisossomo Biogenesis em macrófagos ao vivo

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

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.

Avaliando a inata Sensing de HIV-1 CD4 Infected<sup&gt; +</sup&gt; As células T por células dendríticas plasmocitóides Usando um<em&gt; Ex vivo</em&gt; Co-cultura do sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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

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

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

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

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 ...

Migração fagossomo e velocidade medida no Live primárias macrófagos humanos infectados com o HIV-1

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