Name: in vitro disassembly of influenza a virus capsids by gradient centrifugation
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Description: Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

Agradecemos Yohei Yamauchi e Roberta Mancini por nos fornecer reagentes. O laboratório AH foi apoiada pelas redes de formação inicial Marie Curie (ITN), o Conselho Europeu de Investigação (ERC), e pela Swiss National Science Foundation (Sinergia).

1. Preparação de tampões e soluções de Stock <ol><li> Preparar tampão de MNT (MES 20 mM, Tris 30 mM e NaCl 100 mM) por dissolução de 470 mg de Tris, cloridrato de 390 mg de hidrato de MES e 580 mg de NaCl em 80 ml ​​de ddH 2 O. Ajustar o tampão a pH 7,4 e trazê-lo para um volume final de 100 ml com ddH 2 O. </li><li> Preparar três soluções idênticas de tampão 500 mM MES dissolvendo 9,76 g MES hidratado em 80 ml ​​de dH2O cada um. Ajustar as memórias intermédias para um pH de 5,8, 5,4, e 5,0, respectivamente, por titulação com NaOH concentrado. Traga cada tampão para um volume final de 100 mL com dH2O </li><li> Preparar duas soluções idênticas de tampão Tris 500 mM por dissolução de 7,88 g de Tris cloridrato em 80 ml ​​de dH2O cada e ajustar o pH para 7,4 e 6,4, respectivamente, por titulação com HCl concentrado. Traga o tampão para um volume final de 100 mL com dH2O </li><li> Preparar a solução de glicerol a 50% em dH 2 O e agita-se bem até a GlycErol é homogeneamente misturada. Filtrar a solução através de uma unidade de filtro Steritop (0,22 de tamanho de poro) para um frasco de vidro. </li><li> Preparar solução a 10% de NP-40 e NaCl 1 M em dH2O </li><li> Prepare 25x inibidor de protease concentrada por dissolução de dois comprimidos em 4 ml DDH 2 O. </li></ol> 2. Preparação de gradientes de glicerol <ol><li> Prepare 5 ml de tampão detergente de mistura principal solução para cada condição de pH (300 mM de NaCl, 2% de NP-40, inibidores da protease 2x concentrado) de teste (pH 7.4, 6.4, 5.8, 5.4, e 5.0). Para este efeito, mistura 9 ml de NaCl 1 M, 6 ml de 10% de NP-40, 2,4 ml de solução stock de inibidor de protease, 25x e 9 ml de ddH 2 0. </li><li> Para cada condição pipeta pH 4,4 ml da mistura principal em um tubo de 50 ml. </li><li> Para valores de pH acima de 5,8 adicionar 0,6 ml da respectiva solução de estoque de 500 mM de Tris com pH ajustado ao tubo. Para pH 5,8 e inferior adicionar 0,6 ml da respectiva solução de MES 500 mM com o pH ajustado. </li><li> Faça 5ml de solução tampão contendo NaCl 300 mM, inibidor de protease de 2x, Tris 60 mM ajustado para pH 7,4 e ddH 2 O. Isto irá servir como tampão de gradiente de controlo sem detergente. </li><li> Se necessário, ajustar com precisão o pH das soluções para pH 7,4, 6,4, 5,8, 5,4, e 5,0 por adição de soluções de HCI ou NaOH concentrado, respectivamente. </li><li> Adicionar 5 ml de solução stock de glicerol a 50% e 5 ml de as misturas tampão contendo detergente e sem detergente, resultando em seis diferentes soluções de glicerol a 25%. Verifique os valores de pH utilizando papel indicador de pH. NOTA: No caso de o pH medido difere significativamente do valor desejado, as respectivas soluções devem ser preparadas novamente. </li><li> Preparar solução de glicerol a 15% por mistura de estoque de glicerol a 50% com dH 2 O em uma proporção de 3: 7. </li></ol> 3. Ultracentrifugação da IAV <ol><li> Adicionar solução de glicerol a 3 ml de 15% em tubos de centrifugação ultra-claros (13,2 ml, 14 mm x 89 mm) usando uma seringa de 5 ml e uma needle (21 G, 9 cm de comprimento). Não deixar gotas para a parede interior do tubo como este poderia perturbar a integridade do gradiente. Repetir este para um total de seis tubos de centrifugação, uma para cada um dos cinco condições de pH para testar e uma para a amostra de controlo. </li><li> Com cuidado, coloque solução de glicerol a 3,4 ml de 25% sob a camada de glicerol 15%, utilizando uma seringa de 5 ml e uma agulha longa. Tome cuidado para não misturar as duas camadas. Repita este procedimento para todas as seis condições para testar com as respectivas soluções de glicerol com o pH ajustado. NOTA: Trabalho na classe II biossegurança gabinete para as etapas seguintes. </li><li> Suavemente sobrepor os gradientes de glicerol com 30 ul clarificadas fluido alantóide que contém IAV (X31, H3N2) diluído em 1 ml de tampão de MNT (corresponde a cerca de 20-30 ug da proteína virai total) para cada gradiente. </li><li> Equilíbrio oposição tubos e colocá-los em um rotor SW41 ultracentrifugação. Centrifugar durante 150 minutos, a 55000 x g, e 12 ° C. </li><li> Após a centrifugação, cuidadosamenteremover o sobrenadante, isto é, ambas as camadas de glicerol, usando uma pipeta de Pasteur limpa e um aspirador. Ressuspender o sedimento em 40 ul (1x) não redutoras tampão de amostra LDS. É importante para pipetar para cima e para baixo várias vezes para dissolver completamente o sedimento. Transferir a amostra para um tubo de microcentrífuga de 1,5 ml. </li></ol> 4. SDS-PAGE de fracções de pellets e coloração com Coomassie <ol><li> Aquecer todas as amostras a 95 ° C durante 10 min. Neste ponto, as amostras podem ser armazenadas a -20 ° C até serem analisadas por SDS-PAGE. </li><li> Carga de 20 ul das pelotas dissolvidas em um gradiente de pré-molde (4-12%) em gel de mini-Bis-Tris e correr durante 1 h a 200 V em MOPS SDS 1x tampão de corrida. </li><li> Fazer a solução de fixação com 40% de metanol e ácido acético glacial a 10% em DDH 2 O. </li><li> Incubar o gel na solução de fixação durante 1 hora e manchar durante a noite numa placa de cultura celular 15 cm, com um volume suficiente de solução de Coomassie coloidal enquanto suavementeagitação à temperatura ambiente. NOTA: É importante para fechar o prato, a fim de evitar a evaporação da solução de coloração. </li><li> Destain o gel em ddH 2 O. Substitua o DDH 2 O a cada 15-20 minutos até que o fundo gel torna-se clara. Armazenar o gel em ddH2O a 4 ° C até ser digitalizada para quantificação banda. </li><li> Digitalizar o gel em alta resolução e use um software disponível ou personalizado comercialmente para a quantificação de intensidades das bandas de proteína. Subtrair o sinal de fundo a partir de uma região próxima às respectivas bandas e normalizar estes valores para as amostras de controlo sem detergente (a pH 7,4). </li></ol>

<ol>
	<li>Taubenberger, J. K., Kash, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+Virus+Evolution,+Host+Adaptation,+and+Pandemic+Formation.">Influenza Virus Evolution, Host Adaptation, and Pandemic Formation.</a> Cell host &amp; microbe. 7 (6), 440-451 (2010).</li><li>Skehel, J. J., Wiley, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+binding+and+membrane+fusion+in+virus+entry:+the+influenza+hemagglutinin.">Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.</a> Annual review of biochemistry. 69, 531-569 (2000).</li><li>de Vries, E., 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=Dissection+of+the+influenza+a+virus+endocytic+routes+reveals+macropinocytosis+as+an+alternative+entry+pathway.">Dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway.</a> PLoS pathogens. 7 (3), e1001329 (2011).</li><li>Matlin, K. S., Reggio, H., Helenius, A., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+entry+pathway+of+influenza+virus+in+a+canine+kidney+cell+line.">Infectious entry pathway of influenza virus in a canine kidney cell line.</a> J Cell Biol. 91 (3 Pt 1), 601-613 (1981).</li><li>Patterson, S., Oxford, J. S., Dourmashkin, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+influenza+virus+entry+into+cells.">Studies on the mechanism of influenza virus entry into cells.</a> J Gen Virol. 43 (1), 223-229 (1979).</li><li>Rust, M. J., Lakadamyali, M., Zhang, F., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+endocytic+machinery+around+individual+influenza+viruses+during+viral+entry.">Assembly of endocytic machinery around individual influenza viruses during viral entry.</a> Nat Struct Mol Biol. 11 (6), 567-573 (2004).</li><li>Sieczkarski, S. B., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+virus+entry+via+endocytosis.">Dissecting virus entry via endocytosis.</a> J Gen Virol. 83 (Pt 7), 1535-1545 (2002).</li><li>Yoshimura, A., 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=Infectious+Cell+Entry+Mechanism+of+Influenza+Virus.">Infectious Cell Entry Mechanism of Influenza Virus.</a> Journal of Virology. 43 (1), 284-293 (1982).</li><li>White, J., Kartenbeck, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-fusion+activity+of+influenza+virus.">Membrane-fusion activity of influenza virus.</a> Embo Journal. 1 (2), 217-222 (1982).</li><li>Martin, K., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+transport+of+influenza+virus+ribonucleoproteins:+The+viral+matrix+protein+(M1)+promotes+export+and+inhibits+import.">Nuclear transport of influenza virus ribonucleoproteins: The viral matrix protein (M1) promotes export and inhibits import.</a> Cell. 67 (1), 117-130 (1991).</li><li>Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+47.">Chapter 47.</a> Fields Virology. , 1647-1690 (2006).</li><li>Chou, Y. -. Y., 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=Colocalization+of+Different+Influenza+Viral+RNA+Segments+in+the+Cytoplasm+before+Viral+Budding+as+Shown+by+Single-molecule+Sensitivity+FISH+Analysis.">Colocalization of Different Influenza Viral RNA Segments in the Cytoplasm before Viral Budding as Shown by Single-molecule Sensitivity FISH Analysis.</a> PLoS Pathog. 9 (5), e1003358 (2013).</li><li>Martin, K., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+incoming+influenza+virus+nucleocapsids+into+the+nucleus.">Transport of incoming influenza virus nucleocapsids into the nucleus.</a> Journal of Virology. 65 (1), 232-244 (1991).</li><li>Bui, M., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+M1+protein+and+low+pH+on+nuclear+transport+of+influenza+virus+ribonucleoproteins.">Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins.</a> Journal of Virology. 70 (12), 8391-8401 (1996).</li><li>Li, S., 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=pH-Controlled+Two-Step+Uncoating+of+Influenza+Virus.">pH-Controlled Two-Step Uncoating of Influenza Virus.</a> Biophysical Journal. 106 (7), 1447-1456 (2014).</li><li>Stauffer, S., 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=Stepwise+priming+by+acidic+pH+and+high+K++is+required+for+efficient+uncoating+of+influenza+A+virus+cores+after+penetration.">Stepwise priming by acidic pH and high K+ is required for efficient uncoating of influenza A virus cores after penetration.</a> Journal of Virology. , (2014).</li><li>Zhirnov, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+of+matrix+protein+M1/M+from+virions+occurs+at+different+pH+for+orthomyxo-+and+paramyxoviruses.">Solubilization of matrix protein M1/M from virions occurs at different pH for orthomyxo- and paramyxoviruses.</a> Virology. 176 (1), 274-279 (1990).</li><li>Banerjee, I., 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=Influenza+A+virus+uses+the+aggresome+processing+machinery+for+host+cell+entry.">Influenza A virus uses the aggresome processing machinery for host cell entry.</a> Science. 346 (6208), 473-477 (2014).</li><li>Huotari, J., 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=Cullin-3+regulates+late+endosome+maturation.">Cullin-3 regulates late endosome maturation.</a> Proceedings of the National Academy of Sciences. 109 (3), 823-828 (2012).</li><li>Yamauchi, Y., 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=Histone+Deacetylase+8+Is+Required+for+Centrosome+Cohesion+and+Influenza+A+Virus+Entry.">Histone Deacetylase 8 Is Required for Centrosome Cohesion and Influenza A Virus Entry.</a> PLoS Pathog. 7 (10), e1002316 (2011).</li><li>Bachmayer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Solubilization+of+Hemagglutinin+and+Neuraminidase+from+Influenza+Viruses.">Selective Solubilization of Hemagglutinin and Neuraminidase from Influenza Viruses.</a> Intervirology. 5 (5), 260-272 (1975).</li><li>Reginster, M., Nermut, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Characterization+of+Influenza+Virus+Cores.">Preparation and Characterization of Influenza Virus Cores.</a> Journal of General Virology. 31 (2), 211-220 (1976).</li><li>Nermut, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+Investigation+on+the+Fine+Structure+of+Influenza+Virus.">Further Investigation on the Fine Structure of Influenza Virus.</a> Journal of General Virology. 17 (3), 317-331 (1972).</li><li>Schulze, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+influenza+virus:+II.+A+model+based+on+the+morphology+and+composition+of+subviral+particles.">The structure of influenza virus: II. A model based on the morphology and composition of subviral particles.</a> Virology. 47 (1), 181-196 (1972).</li><li>Candiano, 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=Blue+silver:+A+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25 (9), 1327-1333 (2004).</li><li>Greber, U. F., Singh, I., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+virus+uncoating.">Mechanisms of virus uncoating.</a> Trends in microbiology. 2 (2), 52-56 (1994).</li><li>Yamauchi, Y., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+entry+at+a+glance.">Virus entry at a glance.</a> Journal of Cell Science. 126 (6), 1289-1295 (2013).</li><li>Zhirnov, O. P., Grigoriev, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disassembly+of+Influenza+C+Viruses,+Distinct+from+That+of+Influenza+A+and+B+Viruses+Requires+Neutral-Alkaline+pH.">Disassembly of Influenza C Viruses, Distinct from That of Influenza A and B Viruses Requires Neutral-Alkaline pH.</a> Virology. 200 (1), 284-291 (1994).</li></ol>

The authors have nothing to disclose.

cápsides virais são complexos macromoleculares metaestáveis. Enquanto a montagem de viriões requer a encapsidação e condensação do genoma do vírus, o início da próxima ronda de infecção depende de desmontagem desta estrutura capsídeo compacto. Vírus evoluíram para explorar vários mecanismos celulares para controlar o ciclo de revestimento-desencapsulamento, incluindo receptores celulares, chaperones, enzimas proteolíticas, forças físicas proporcionadas pelas proteínas do motor ou helicases, bem como...

Vírus da Influenza A (IAV) é um vírus envolvido que pertence à família de Orthomyxoviridae. Seus genes são codificados em uma, de sentido negativo e segmentada genoma de ARN de cadeia simples. Nos seres humanos, IAV causa infecções respiratórias, que ocorrem em surtos epidêmicos sazonais e tem o potencial para pandemias globais 1. Após a ligação de resíduos de ácido siálico na superfície das células de acolhimento 2, IAV é internalizado por endocitose dependente de clatrina e vias independentes de clatrina 3-8. O meio ácido (pH &lt;5,5) nos vacúolos de endocitose desencadeia uma mudança conformacional na IAV pico glicoproteína hemaglutinina (HA), o que resulta na fusão do vírus e no final da membrana endossomal 9. Uma vez que a cápside IAV (aqui também referido como &quot;núcleo&quot; viral) escapou de endossomas tardios (EIE), que é não revestidos no citosol seguido por transporte das ribonucleoproteínas virais (vRNPs) no núcleo - o local de Oa replicação do vírus e transcrição F 10-13. Antes da activação de ácido HA do vírus sofre uma redução gradual do pH no sistema endocítico, que prepara o núcleo para a sua desmontagem posterior 14-16. Neste &quot;priming&quot; intensificar os canais de íon M2 na membrana viral mediar o influxo de prótons e K +10,14,16. A mudança na concentração iônica no interior vírus perturba as interações construir pela proteína da matriz viral M1 ea vRNP pacote de oito, e facilita IAV uncoating no citoplasma seguindo a fusão da membrana 10,14-17. análise directa e quantitativa do passo de iniciação tem sido dificultada pelo facto de endossomas são difíceis de aceder experimentalmente. O processo de entrada é, aliás, altamente não-síncrona. Além disso, ensaios de ponto final, como qRT-PCR de RNA ou da infecciosidade medições virais libertados, não fornecem uma imagem detalhada sobre o estado bioquímico do capsi virald em qualquer etapa de entrada. Enquanto perturbação da endossomas por siRNA ou tratamento de drogas tem contribuído significativamente para a compreensão de entrada IAV 18-20, ajuste fino é difícil e propenso a efeitos colaterais não específicos no programa endosome maturação rigidamente controlado. Para evitar estes problemas, nós adaptamos um protocolo previamente desenvolvido in vitro, com base na utilização de centrifugação em gradiente de velocidade de 17. Em contraste com outras tentativas 21,22, 23,24, que eram na maior parte baseados em combinações de clivagem proteolítica e tratamento com detergente, seguido por análise de EM, esta abordagem permitiu um resultado facilmente quantificáveis. Centrifugação através de diferentes camadas de gradiente permite que as partículas sedimentam a ser expostos e reagem com mudança de condições, de forma controlada. No protocolo apresentado, IAV derivados a partir de fluido alantóico clarificado ou partículas virais purificadas são sedimentadas numa glicerol de duas camadasgradiente em que a camada inferior contém o detergente não-iónico NP-40 (Figura 1). Tal como o vírus entra na segunda camada, contendo detergente, o envelope lipídico e glicoproteínas do envelope viral são suavemente solubilizada e deixado para trás. O núcleo, composto pelo feixe vRNP e oito rodeado por uma camada de matriz, sedimentos como uma estrutura estável na fracção de sedimento. proteínas de núcleo virai, tais como M1 e nucleoproteína vRNP-associado (NP), pode ser identificado no sedimento por SDS-PAGE e coloração com Coomassie. Em particular, tomando vantagem de géis de gradiente disponíveis comercialmente e a coloração com Coomassie coloidal altamente sensível 25, permite alta precisão e a detecção de mesmo pequenas quantidades de proteínas associadas ao núcleo virai. Isso define a base para testar se condições diferentes, como pH, concentração de sal, e os fatores não revestidos putativos têm efeitos sobre o comportamento de sedimentação dos componentes do núcleo e stabil núcleo<html> dade. Para este objectivo, apenas a camada inferior, contendo o detergente em o gradiente de glicerol é modificado através da introdução do factor ou condição de interesse. A técnica tem sido particularmente útil em investigar o efeito de diferentes valores de pH e concentrações de sal sobre a integridade do núcleo 16 IAV. Etapas intermediárias de IAV desencapsulamento poderia ser monitorada incluindo dissociação da camada de matriz a um pH ligeiramente ácido (&lt;6,5), seguido de vRNP de dissociação a pH 5,5 e inferior 16,17 (Figura 1). O último passo foi adicionalmente reforçada pela presença de alta concentração de K + na camada de glicerol, reflectindo um ambiente endocítica final 16. Assim, como o vírus e o núcleo sedimentado através do gradiente que experimentaram uma mudança de meio, imitando as condições em endossomas. O resultado foi uma desmontagem gradual do núcleo virai in vitro, completando os resultados derivados a partir de ensaios de biologia celular. t &quot;&gt; O método aqui apresentado, permitiu rápido e altamente reprodutível análise de IAV (X31 e A / WSN / 33) e IBV (B / Lee / 40) desencapsulamento desencadeada pelo pH ácido e aumentando +16,17 K, bem como desmontagem de núcleos paramixovírus após exposição pH alcalino 17. é concebível que a abordagem pode ser adaptada para outros vírus com envelope para obter insights sobre as propriedades bioquímicas da estrutura da cápside virai e desmontagem da cápside durante a entrada na célula.

<table><tbody><tr><td>cOmplete&amp;trade;, EDTA-free protease inhibitor tablets</td><td>Sigma-Aldrich</td><td>11873580001</td><td>The stock solution can be stored at 2 to 8 &amp;deg;C for 1 to 2 weeks</td></tr><tr><td>Glacial acetic acid</td><td>Merck Millipore</td><td>100063</td><td></td></tr><tr><td>Glycerol anhydrous BioChemica</td><td>AppliChem</td><td>A1123</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck Millipore</td><td>100317</td><td></td></tr><tr><td>Long injection needle (21&amp;nbsp;G, 9 cm, bevel or blunt-end)</td><td></td><td></td><td></td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>NP-40</td><td>Sigma-Aldrich</td><td>I8896</td><td>Now commercially available as IGEPAL&amp;reg; CA-630</td></tr><tr><td>NuPAGE 4-12% Bis-Tris mini gels, 10&amp;nbsp;wells, 1.0 mm</td><td>Life Technologies</td><td>NP0321</td><td></td></tr><tr><td>NuPAGE LDS sample buffer (4x)</td><td>Life Technologies</td><td>NP0008</td><td></td></tr><tr><td>NuPAGE MOPS SDS running buffer (20x)</td><td>Life Technologies</td><td>NP0001</td><td></td></tr><tr><td>pH indicator strips, pH 4.0-7.0</td><td>Merck Millipore</td><td>109542</td><td></td></tr><tr><td>QC Colloidal Coomassie Stain</td><td>BIO RAD</td><td>1610803</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>106406</td><td></td></tr><tr><td>Sodiumhydroxide</td><td>Merck Millipore</td><td>106498</td><td></td></tr><tr><td>Steritop filter unit</td><td>Merck Millipore</td><td>SCGPT05RE</td><td></td></tr><tr><td>SW41 Ti, ultracentrifuge rotor set</td><td>Beckman Coulter</td><td>331336</td><td></td></tr><tr><td>Thinwall, Ultra-clear centrifuge tubes, 13.2&amp;nbsp;ml, 14 mm x 89 mm</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Tris hydrochloride&amp;nbsp;</td><td>AppliChem</td><td>A1087</td><td></td></tr><tr><td>X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid</td><td>Virapur</td><td></td><td>Freshly thawed on 4 &amp;deg;C</td></tr></tbody></table>

in vitro disassembly of influenza a virus capsids by gradient centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

Como já foi discutido, priming em endossomos é necessária para tornar o núcleo IAV perda do revestimento competente. A protonação do núcleo enfraquece interacção de M1 e vRNPs (composto do RNA viral, NP, e o complexo de polimerase PB1 / PB2 / PA). Este processo inicia-se quando o vírus de entrada está exposta a um pH de 6.5 (ou inferior) em endossomas precoces (EES) e continua até que os fusíveis de vírus em torno de pH 5,0 em les. <p class="jove_content" fo:keep-togeth...

Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In Vitro A desmontagem do vírus influenza A capsï¿½eos por centrifugação em gradiente

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

in-vitro-disassembly-influenza-virus-capsids-gradient

Research

JoVE Journal

Immunology and Infection

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

8.9K visualizações.  ETH Zurich. Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Vídeo: In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Preparação de DNA viral a partir nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Imunologia e Infecção

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 ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

Simples e robusto In vivo E In vitro Abordagem para o Estudo Assembléia Virus

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Purificação por afinidade de Complexos de Influenza Virus ribonucleoproteína da cromatina de células infectadas

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

Purificação e Visualização de Influenza A complexos de ribonucleoproteínas Viral

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Biologia

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Métodos de alternativas In Vitro para a determinação da integridade estrutural do capsídeo Viral

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

Montagem e purificação do protótipo vírus espumoso Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Um procedimento simplificado e padronizado para gerar vetores de vírus adeno-associados de alto título e alta qualidade utilizando uma plataforma de fábrica de células

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

Bioengenharia

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Micro-aspiração de célula única como uma estratégia alternativa à classificação celular ativada por fluorescência para separação de mistura de vírus gigante

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Isolamento de AAV por centrifugação de gradiente de densidade de iodixanol duplo

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Autor em destaque: Isolamento eficiente de vírus adeno-associados para aplicações pré-clínicas

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

Produção e purificação de vírus adenoassociados em cultura de suspensão por centrifugação por gradiente de densidade de iodixanol para aplicações in vivo