Name: in vitro disassembly of influenza a virus capsids by gradient centrifugation
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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...

<ol>
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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 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.

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		<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 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...

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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 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 ...

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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