We thank Yohei Yamauchi and Roberta Mancini for providing us with reagents. The A. H. laboratory was supported by the Marie Curie Initial Training Networks (ITN), the European Research Council (ERC), and by the Swiss National Science Foundation (Sinergia).

1. Preparation of Buffers and Stock Solutions
<ol>
	<li>Prepare MNT buffer (20 mM MES, 30 mM Tris and 100 mM NaCl) by dissolving 470 mg Tris Hydrochloride, 390 mg MES hydrate and 580 mg NaCl in 80 ml ddH2O. Adjust the buffer to pH 7.4 and bring it to a final volume of 100 ml with ddH2O.</li>
	<li>Prepare three identical solutions of 500 mM MES buffer by dissolving 9.76 g MES hydrate in 80 ml dH2O each. Adjust the buffers to pH 5.8, 5.4, and 5.0, respectively, by titrating with concentra...

<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>

Viral capsids are metastable macromolecular complexes. While assembly of virions requires the encapsidation and condensation of the virus genome, initiation of the next round of infection depends on disassembly of this compact capsid structure. Viruses have evolved to exploit various cellular mechanisms to control the coating-uncoating cycle, including cellular receptors, chaperones, proteolytic enzymes, physical forces provided by motor proteins or helicases as well as pH and ionic switches26,27. Here, we des...

Influenza A virus (IAV) is an enveloped virus and belongs to the family of Orthomyxoviridae. Its genes are encoded on a segmented, negative-sense and single-stranded RNA genome. In humans, IAV causes respiratory infections, which occur in seasonal epidemic outbreaks and bears the potential for global pandemics1. Upon binding to sialic acid residues on the host cell surface2, IAV is internalized by clathrin-dependent endocytosis and clathrin-independent pathways3-8. The acidic milieu (pH &#60; 5.5) in the endocytic vacuoles triggers a major conformational change in the IAV spike glycoprotein hemagglutinin (HA), which results in fusion of the viral and the late endosomal membrane9. Once the IAV capsid (here also referred to as viral &#34;core&#34;) has escaped from late endosomes (LEs), it is uncoated in the cytosol followed by transport of the viral ribonucleoproteins (vRNPs) into the nucleus &#8212; the site of virus replication and transcription10-13. Prior to acid activation of HA the virus experiences a gradual decrease in pH in the endocytic system, which primes the core for its subsequent disassembly14-16. In this &#34;priming&#34; step the M2 ion channels in the viral membrane mediate the influx of protons and K+10,14,16. The change in ionic concentration in the virus interior disturbs the interactions build up by the viral matrix protein M1 and the eight vRNP bundle, and facilitates IAV uncoating in the cytoplasm following membrane fusion10,14-17.
Direct and quantitative analysis of the priming step has been hampered by the fact that endosomes are difficult to access experimentally. The entry process is, moreover, highly non-synchronous. In addition, end-point assays, such as qRT-PCR of released viral RNA or infectivity measurements, do not provide a detailed picture about the biochemical state of the viral capsid at any given step of entry. While perturbation of endosomes by siRNA or drug treatment has significantly contributed to the understanding of IAV entry18-20, fine-tuning is difficult and prone to unspecific side effects in the tightly controlled endosome maturation program.
To avoid these problems, we have adapted a previously developed in vitro protocol based on the use of velocity gradient centrifugation17. In contrast to other attempts 21,22,23,24, which were mostly based on combinations of proteolytic cleavage and detergent treatment followed by EM analysis, this approach enabled an easily quantifiable result. Centrifugation through different gradient layers allows the sedimenting particles to be exposed to and react with changing conditions in a controlled manner. In the presented protocol, IAV derived from clarified allantoic fluid or purified viral particles are sedimented into a two-layer glycerol gradient in which the bottom layer contains the non-ionic detergent NP-40 (Figure 1). As the virus enters the second, detergent-containing layer, the viral lipid envelope and envelope glycoproteins are gently solubilized and left behind. The core, composed of the eight vRNP bundle and surrounded by a matrix layer, sediments as a stable structure into the pellet fraction. Viral core proteins, such as M1 and vRNP-associated nucleoprotein (NP), can be identified in the pellet by SDS-PAGE and Coomassie staining. In particular, taking advantage of commercially available gradient gels and staining with the highly sensitive colloidal Coomassie25, enables high precision and detection of even small amounts of viral core-associated proteins.
This sets the basis for testing whether different conditions such as pH, salt concentration, and putative uncoating factors have effects on the sedimentation behavior of core components and core stability. To this aim, only the lower, detergent-containing layer in the glycerol gradient is modified by introducing the factor or condition of interest. The technique has been particularly valuable in investigating the effect of different pH values and salt concentrations on the integrity of the IAV core16. Intermediate steps of IAV uncoating could be monitored including dissociation of the matrix layer at mildly acidic pH (&#60;6.5) followed by vRNP dissociation at pH 5.5 and lower16,17 (Figure 1). The latter step was further enhanced by the presence of high K+ concentration in the glycerol layer, reflecting a late endocytic environment16. Thus, as the virus and the core sedimented through the gradient they experienced a changing milieu that mimics conditions in endosomes. The outcome was a stepwise disassembly of the viral core in vitro, complementing results derived from cell biology assays.
The method presented here has enabled fast and highly reproducible analysis of IAV (X31 and A/WSN/33) and IBV (B/Lee/40) uncoating triggered by acidic pH and increasing K+16,17 as well as disassembly of paramyxovirus cores upon alkaline pH exposure17. It is conceivable that the approach can be adapted to other enveloped viruses to gain insights into the biochemical properties of the viral capsid structure and capsid disassembly during cell entry.

<table border="0" cellpadding="0" cellspacing="0" width="1103">
	
	<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>
</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.

As already discussed, priming in endosomes is required to render the IAV core uncoating competent. Protonation of the core weakens interaction of M1 and vRNPs (composed of the viral RNA, NP, and the polymerase complex PB1/PB2/PA). This process is initiated when incoming virus is exposed to a pH of 6.5 (or lower) in early endosomes (EEs) and continues until the virus fuses at around pH 5.0 in LEs.
In order to mimic the decrease o...

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 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 ...

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.

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

Research

JoVE Journal

Immunology and Infection

8.8K vistas.  ETH Zurich. 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 u...