Name: in vitro disassembly of influenza a virus capsids by gradient centrifugation
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Agradecemos a Yohei Yamauchi y Roberta Mancini para que nos proporciona reactivos. El laboratorio AH fue apoyada por las Redes de formación inicial Marie Curie (ITN), el Consejo Europeo de Investigación (CEI), y por la Fundación Nacional de Ciencia de Suiza (Sinergia).

1. Preparación de tampones y soluciones de archivo <ol><li> Preparar tampón SM (MES 20 mM, Tris 30 mM y NaCl 100 mM) disolviendo 470 mg de clorhidrato de Tris, 390 mg de hidrato de MES y 580 mg de NaCl en 80 ml ​​ddH2O Ajustar el tampón a pH 7,4 y llevarlo a un volumen final de 100 ml con ddH 2 O. </li><li> Preparar tres soluciones idénticas de tampón MES 500 mM disolviendo 9,76 g de hidratos de MES en 80 ml ​​de H2O destilada cada uno. Ajuste los tampones a pH 5,8, 5,4, y...

<ol>
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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. 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cápsides virales son complejos macromoleculares metaestables. Durante el montaje de los viriones requiere la encapsidación y la condensación del genoma del virus, el inicio de la siguiente ronda de infección depende de desmontaje de esta estructura de la cápside compacto. Los virus han evolucionado para explotar varios mecanismos celulares para controlar el ciclo de recubrimiento-uncoating, incluyendo receptores celulares, chaperones, enzimas proteolíticas, fuerzas físicas proporcionadas por proteínas motoras o ...

Influenza A virus (IAV) es un virus con envoltura y pertenece a la familia de Orthomyxoviridae. Sus genes están codificados en un sentido negativo segmentado y genoma de ARN de cadena simple. En los seres humanos, IAV causa infecciones respiratorias, que se producen en brotes epidémicos estacionales y tiene el potencial de pandemias globales 1. Tras la unión a los residuos de ácido siálico en la superficie de la célula huésped 2, IAV es internalizado por endocitosis dependiente de clatrina y las vías de clathrin independiente 3-8. El medio ácido (pH &lt;5,5) en las vacuolas endocíticas desencadena un cambio conformacional importante en la IAV espiga glicoproteína hemaglutinina (HA), lo que resulta en la fusión de la viral y el fallecido membrana 9 endosomal. Una vez que la cápside IAV (aquí también se hace referencia como &quot;núcleo&quot; viral) ha escapado de los endosomas finales (LES), que no está recubierto en el citosol seguido por el transporte de las ribonucleoproteínas virales (vRNPs) en el núcleo - el sitio ola replicación del virus y la transcripción f 10-13. Antes de la activación ácida de HA del virus experimenta una disminución gradual en el pH en el sistema endocítica, que ceba el núcleo para su posterior desmontaje 14-16. En este &quot;cebado&quot; intensificar los canales iónicos M2 en la membrana viral mediar en la afluencia de protones y K +10,14,16. El cambio en la concentración iónica en el interior virus perturba las interacciones se acumulan por la proteína de la matriz M1 viral y el haz vRNP ocho, y facilita IAV uncoating en el citoplasma después de la fusión de membranas 10,14-17. El análisis directo y cuantitativa de la etapa de cebado se ha visto obstaculizada por el hecho de que los endosomas son difíciles de acceder experimentalmente. El proceso de entrada es, por lo demás, altamente no sincrónico. Además, los ensayos de punto final, como QRT-PCR mediciones de ARN o la infectividad viral liberados, no proporcionan una imagen detallada sobre el estado bioquímico de la capsi virald en cualquier etapa dada de entrada. Mientras que la perturbación de los endosomas de siRNA o tratamiento de drogas ha contribuido significativamente a la comprensión de la entrada IAV 18-20, puesta a punto es difícil y propensos a los efectos secundarios inespecíficos en el programa endosoma maduración estrechamente controlada. Para evitar estos problemas, hemos adaptado un protocolo previamente desarrollado in vitro basado en el uso de la velocidad de centrifugación en gradiente de 17. A diferencia de otros intentos 21,22, 23,24, que se basan principalmente en combinaciones de escisión proteolítica y tratamiento con detergente seguido por análisis de EM, este enfoque permitió un resultado fácilmente cuantificable. La centrifugación a través de diferentes capas de gradiente permite que las partículas que sedimentan a estar expuestos a y reaccionan con las condiciones cambiantes de una manera controlada. En el protocolo presentado, IAV derivado de fluido alantoideo aclarado o partículas virales purificadas se sedimentan en un glicerol de dos capasgradiente en el que la capa inferior contiene el detergente no iónico NP-40 (Figura 1). A medida que el virus entra en la segunda capa, que contiene detergente, la envoltura de lípidos y glicoproteínas de la envoltura viral se solubilizan con suavidad y dejaron atrás. El núcleo, formado por el haz de ocho vRNP y rodeado por una capa de matriz, los sedimentos como una estructura estable en la fracción de sedimento. proteínas del núcleo viral, tales como M1 y nucleoproteína asociado-vRNP (NP), se pueden identificar en el sedimento por SDS-PAGE y tinción Coomassie. En particular, tomando ventaja de geles de gradiente disponibles en el mercado y la tinción con el coloidal Coomassie altamente sensible 25, permite una alta precisión y la detección de incluso pequeñas cantidades de proteínas del núcleo asociada virales. Esto establece la base para probar si las diferentes condiciones tales como el pH, la concentración de sal, y los factores putativos Uncoating tener efectos sobre el comportamiento de sedimentación de los componentes del núcleo y de núcleo Stabil<html> dad. Para este objetivo, solamente la capa que contiene detergente menor en el gradiente de glicerol se modifica mediante la introducción del factor o condición de interés. La técnica ha sido particularmente valiosa en la investigación del efecto de diferentes valores de pH y concentraciones de sal en la integridad del núcleo 16 IAV. Pasos intermedios de IAV uncoating podrían ser monitorizados incluyendo la disociación de la capa de la matriz a pH ligeramente ácido (&lt;6,5), seguido de vRNP disociación a pH 5,5 e inferior 16,17 (Figura 1). Este último paso se ha mejorado aún más por la presencia de una alta concentración de K + en la capa de glicerol, lo que refleja un entorno endocítica finales 16. Por lo tanto, como el virus y el núcleo sedimentan a través del gradiente que experimentaron un medio de cambio que imita las condiciones en los endosomas. El resultado fue un desmontaje por etapas del núcleo viral in vitro, como complemento de los resultados derivados de los ensayos de biología celular. t &quot;&gt; El método que aquí se presenta ha permitido el análisis rápido y altamente reproducible de IAV (X31 y A / WSN / 33) e IBV (B / Lee / 40) Uncoating desencadenada por pH ácido y el aumento de K +16,17, así como el desmontaje de núcleos de paramixovirus tras la exposición pH alcalino 17. es concebible que el enfoque se puede adaptar a otros virus con envoltura para obtener información sobre las propiedades bioquímicas de la estructura de la cápside viral y desmontaje de la cápside durante la entrada en la célula.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">cOmplete&#8482;, EDTA-free protease inhibitor tablets</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:116px;">11873580001</td>
			<td style="width:473px;">The stock solution can be stored at 2 to 8 &#176;C for 1 to 2 weeks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Merck Millipore</td>
			<td>100063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol anhydrous BioChemica</td>
			<td>AppliChem</td>
			<td>A1123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Merck Millipore</td>
			<td>100317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Long injection needle (21 GA, 9 cm, bevel or blunt-end)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td>Now commercially available as IGEPAL&#174; CA-630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; 4-12% Bis-Tris mini gels, 10 wells, 1.0 mm</td>
			<td>Life Technologies</td>
			<td>NP0321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; LDS sample buffer (4X)</td>
			<td>Life Technologies</td>
			<td>NP0008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE&#174; MOPS SDS running buffer (20X)</td>
			<td>Life Technologies</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH indicator strips, pH 4.0 - 7.0</td>
			<td>Merck Millipore</td>
			<td>109542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QC Colloidal Coomassie Stain</td>
			<td>BIO RAD</td>
			<td>1610803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Merck Millipore</td>
			<td>106406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodiumhydroxide</td>
			<td>Merck Millipore</td>
			<td>106498</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Steritop&#8482; filter unit</td>
			<td>Merck Millipore</td>
			<td>SCGPT05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW41 Ti, ultracentrifuge rotor set</td>
			<td>Beckman Coulter</td>
			<td>331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thinwall, Ultra-clear centrifuge tubes, 13.2 ml, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris hydrochloride&#160;</td>
			<td>AppliChem</td>
			<td>A1087</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid</td>
			<td>Virapur</td>
			<td></td>
			<td>Freshly thawed on 4&#176;C</td>
		</tr>
	</tbody>
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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 ya se ha comentado, el cebado en los endosomas que se requiere para hacer que el núcleo IAV uncoating competente. La protonación del núcleo debilita la interacción de M1 y vRNPs (compuesto del ARN viral, NP, y la polimerasa PB1 complejo / PB2 / PA). Este proceso se inicia cuando el virus entrante se expone a un pH de 6,5 (o inferior) en los endosomas tempranos (EEE) y continúa hasta que los fusibles de virus en torno a pH 5,0 en LES. <p class="jove_content" fo:keep-together...

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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 El desmontaje de virus de influenza A cápsides por centrifugación en 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 ...

The overall goal of this velocity gradient centrifugation is to analyze influenza A virus capsids under conditions mimicking the endocytic milieu that incoming viruses encounter during cell entry. This method can help answer key questions about how influenza A virus capsids disassemble during cell entry, such as the specific effect of mildly acidic pH on capsid stability. The main advantage of this technique is that it enables quantitative analysis of the influenza A virus uncoating process under defined experimental conditions.Besides providing insights into how influenza A virus uncoating is initiated, this method can also be applied to the study other envelope viruses with related uncoating mechanisms. Demonstrating the procedure, will be Firat Nebloglu, a former student from my lab. After preparing stock buffers and reagents according to the text protocol, for each pH condition to test, prepare detergent buffer master mix by mixing nine milliliters of one Molar sodium chloride, six milliliters of 10%NP40, 2.4 milliliters of the 25X protease inhibitor stock, and nine milliliters of doubly-distilled water.Then for each pH condition, pipet 4.4 milliliters of the master mix into a 50 milliliter conical tube. For pH values above 5.8, add 0.6 milliliters of the respective pH-adjusted 500 milliMolar Tris stock solution to the tube. For pH values 5.8 and lower, add 0.6 milliliters of the respective pH-adjusted 500 milliMolar MES stock solution.Make five milliliters of buffer solution, containing 300 milliMolar sodium chloride, 2X protease inhibitor, 60 milliMolar Tris, adjusted to pH 7.4, and double-distilled water. This will serve as the detergent-free control gradient buffer. Next, add five milliliters of the 50%glycerol stock to five milliliters of the detergent-containing and detergent-free buffer mixtures, resulting six different 25%glycerol solutions.Use pH strips to verify the pH values. Prepare a 15%glycerol solution by mixing a three to seven ratio of 50%glycerol stock with distilled water. Using a five milliliter syringe and a needle, add three milliliters of 15%glycerol solution to six ultraclear centrifugation tubes.Wash the needle in double-distilled water after every step. Use a five milliliter syringe and a long needle to carefully inject 3.4 milliliters of 25%glycerol solution under the 15%glycerol layer, taking care not to mix the two layers. An interface between both layers is clearly visible.Repeat this for all six conditions to test with the respective pH-adjusted glycerol solutions. Next, in a Class II biosafety cabinet, use one milliliter of clarified, diluted allantoic fluid, containing influenza A virus, to gently overlay the glycerol gradients. Transfer tubes into rotor buckets, balance opposing tubes, and carefully place them into a pre-cooled SW-41 ultracentrifugation rotor.Centrifuge at 2100 rpm and 12 degrees Celsius for 150 minutes. After the centrifugation, use a clean Pasteur pipette and an aspirator to carefully remove both glycerol layers. Then with 40 milliliters of 1X non-reducing LDS sample buffer, completely resuspend the pellet by pipetting up and down before transferring the sample into a 1.5 milliliter microcentrifuge tube.To carry out SDS-PAGE, heat all the samples at 95 degrees Celsius for 10 minutes. Load 20 microliters of the dissolved pellets onto a precast gradient Bis-Tris mini-gel and run at 200 Volts for one hour in 1X MOPS SDS running buffer. Incubate the gel in fixation solution for one hour.Rinse one to two times with water, and stain overnight in a 15 centimeter cell culture dish with a sufficient volume of colloidal Coomassie solution while gently shaking at room temperature. Destain the gel in doubly-distilled water, replacing the liquid every 15 to 20 minutes until the gel background becomes clear. Store the gel in doubly-distilled water at four degrees Celsius until it is scanned for band quantification.Finally, scan the gel at high resolution and use commercially available custom-made software for quantification of protein band intensities. Subtract the background signal from a region close to the respective bands and normalize the values to the detergent-free control values. In this experiment, X31 derived from clarified allantoic fluid was subjected to velocity gradient centrifugation at pH levels between 7.4 and 5.0 and analyzed by SDS-PAGE.Without detergent present in the gradient, intact virions are pelleted as reflected by the characteristic pattern of bands, representing HA, NP, and M1 in the Coomassie-stained gel. Upon addition of NP40 to the bottom glycerol layer, the viral envelope, including HA, NA, and M2, was solubilized and the viral core alone sedimented into the pellet during ultracentrifugation. Starting at a pH below 6.5, M1 is gradually lost from the pellet fraction, reaching a minimum between pH 5.8 and pH 5.4.Below pH 5.8, vRNPs were dissociated and thus lost from the pellet. From the protein bands analyzed, two distinct pH thresholds for disassembly of the M1 layer and dissociation of vRNP bundles were revealed, including pH 6.5 and pH 5.8, respectively. While attempting this procedure, it's important to remember that depending on the particular influenza A virus strain or mutant, different disassembly properties may be observed.Following this procedure, other methods like electron microscopy can be performed in order to gain additional insights like the structure of isolated influenza virus capsids.

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Immunology and Infection

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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