Name: in vitro disassembly of influenza a virus capsids by gradient centrifugation
Uploaded: 2016-03-27T15:00:00.000+00:00
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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

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 concentrated NaOH. Bring each buffer to a final volume of 100 ml with dH2O.</li>
	<li>Prepare two identical solutions of 500 mM Tris buffer by dissolving 7.88 g Tris Hydrochloride in 80 ml dH2O each and adjust the pH to 7.4 and 6.4, respectively, by titrating with concentrated HCI. Bring the buffer to a final volume of 100 ml with dH2O.</li>
	<li>Prepare 50% glycerol solution in dH2O and stir well until the glycerol is homogenously mixed. Filter the solution through a Steritop filter unit (0.22 &#181;m pore size) into a glass bottle.</li>
	<li>Prepare 10% NP-40 solution and 1 M NaCl in dH2O.</li>
	<li>Prepare 25x concentrated protease inhibitor by dissolving two tablets in 4 ml ddH2O.</li>
</ol>
2. Preparation of Glycerol Gradients
<ol>
	<li>Prepare 5 ml detergent buffer master mix (300 mM NaCl, 2% NP-40, 2x concentrated protease inhibitor) solution for each pH condition to test (pH 7.4, 6.4, 5.8, 5.4, and 5.0). For this purpose, mix 9 ml of 1 M NaCl, 6 ml of 10% NP-40, 2.4 ml of the 25x protease inhibitor stock, and 9 ml ddH20.</li>
	<li>For each pH condition pipet 4.4 ml of the master mix into a 50 ml conical tube.</li>
	<li>For pH values above 5.8 add 0.6 ml of the respective pH-adjusted 500 mM Tris stock solution to the tube. For pH 5.8 and lower add 0.6 ml of the respective pH-adjusted 500 mM MES stock solution.</li>
	<li>Make 5 ml of buffer solution containing 300 mM NaCl, 2x protease inhibitor, 60 mM Tris adjusted to pH 7.4 and ddH2O. This will serve as the detergent-free control gradient buffer.</li>
	<li>If necessary, fine-adjust the pH of the solutions to pH 7.4, 6.4, 5.8, 5.4, and 5.0 by adding concentrated HCI or NaOH solutions, respectively.</li>
	<li>Add 5 ml of the 50% glycerol stock to 5 ml of the detergent-containing and detergent-free buffer mixtures resulting in six different 25% glycerol solutions. Verify the pH values by using pH indicator strips. 
	NOTE: In case the measured pH differs significantly from the desired value, the respective solutions should be prepared again.</li>
	<li>Prepare 15% glycerol solution by mixing 50% glycerol stock with dH2O in a 3:7 ratio.</li>
</ol>
3. Ultracentrifugation of IAV
<ol>
	<li>Add 3 ml 15% glycerol solution into ultra-clear centrifugation tubes (13.2 ml, 14 mm x 89 mm) by using a 5 ml syringe and a needle (21 G, 9 cm long). Do not leave drops at the inner wall of the tube as this might disturb the integrity of the gradient. Repeat this for a total of six centrifugation tubes, one for each of the five pH conditions to test and one for the control sample.</li>
	<li>Carefully place 3.4 ml 25% glycerol solution under the 15% glycerol layer by using a 5 ml syringe and a long needle. Take care to not mix the two layers. Repeat this for all six conditions to test with the respective pH-adjusted glycerol solutions. 
	NOTE: Work in class II biosafety cabinet for the following steps.</li>
	<li>Gently overlay the glycerol gradients with 30 &#181;l clarified allantoic fluid containing IAV (X31, H3N2) diluted in 1 ml MNT buffer (corresponds to around 20-30 &#181;g of total viral protein) for each gradient.</li>
	<li>Balance opposing tubes and place them into a SW41 ultracentrifugation rotor. Centrifuge for 150 min, at 55,000 x g, and 12 &#176;C.</li>
	<li>After the centrifugation, carefully remove the supernatant, i.e., both glycerol layers by using a clean Pasteur pipette and an aspirator. Resuspend the pellet in 40 &#181;l (1x) non-reducing LDS sample buffer. It is important to pipet up and down several times to dissolve the pellet completely. Transfer the sample into a 1.5 ml microcentrifuge tube.</li>
</ol>
4. SDS-PAGE of Pellet Fractions and Coomassie Staining
<ol>
	<li>Heat all samples at 95 &#176;C for 10 min. At this point the samples could be stored at -20 &#176;C until they are analyzed by SDS-PAGE.</li>
	<li>Load 20 &#181;l of the dissolved pellets onto a pre-cast gradient (4-12%) Bis-Tris mini gel and run for 1 hr at 200 V in 1x MOPS SDS running buffer.</li>
	<li>Make fixation solution with 40% methanol and 10% glacial acetic acid in ddH2O.</li>
	<li>Incubate the gel in fixation solution for 1 hr and stain overnight in a 15 cm cell culture dish with a sufficient volume of colloidal Coomassie solution while gently shaking at room temperature. 
	NOTE: It is important to close the dish in order to avoid evaporation of the staining solution.</li>
	<li>Destain the gel in ddH2O. Replace the ddH2O every 15-20 min until the gel background becomes clear. Store the gel in ddH2O at 4 &#176;C until it is scanned for band quantification.</li>
	<li>Scan the gel at high resolution and use commercially available or custom-made software for quantification of protein band intensities. Subtract the background signal from a region close to the respective bands and normalize these values to the detergent-free control samples (at pH 7.4).</li>
</ol>

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The authors have nothing to disclose.

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 describe an in vitro approach based on a glycerol gradient centrifugation to specifically test the effect of factors promoting disassembly of the IAV core. The technique can be potentially adapted for dissecting uncoating processes of other enveloped viruses.
By using colloidal Coomassie, the major structural IAV proteins M1, NP, and HA can be clearly detected in SDS-PAGE even with a relatively small virus particle number. Yet, a more in-depth characterization of the sedimented capsid structure and its composition would require follow-up analyses, such as electron microscopy (as previously presented28), Western blotting, dynamic light scattering or mass spectrometry. Further extension of the protocol could include fractionation of the lower (25%) glycerol phase and analysis of the specific core components.
Adjustment of the lower glycerol layer to the optimal fusion pH of 5.0 (for X31) led to an almost complete dissociation of the matrix layer (Figure 2)16. However, around 30% of the input NP signal can still be detected at this pH. It is possible that due to the absence of cellular uncoating factors in this setup, such as recently identified histone deacetylase 6 (HDAC6)18, incomplete disassembly occurs. Inside the cell, slow acidification of the viral core and exposure to a switch from Na+ to K+ primes the virus for complete vRNP release controlled by uncoating factors16. We cannot exclude that in our setup, the non-ionic detergent partially disrupts the acid-exposed and destabilized core. However, no effect on the core sedimenting at neutral pH is observed indicated by similar protein band intensities as compared to non-solubilized samples (Figure 2A, compare lanes 1 and 2). Although, the assay presented here proved to be highly reproducible and (as such is) robust enough for band quantification, a certain range of variation could be observed.
The use of glycerol in this protocol is critical for the outcome of the experiment. As previously reported17, ultracentrifugation through a sucrose gradient destabilizes the IAV core, which is likely due to a high osmotic force created by sucrose. Purification of IAV via sucrose gradients might exert similar effects. Therefore we compared egg-grown X31 derived from clarified allantoic fluid and sucrose-purified stocks. No major difference could be observed with respect to the influence of acidic pH on core stability (data not shown). Nevertheless, in order to exclude potential side effects of the osmotically active sucrose, we recommend performing the in vitro uncoating assay with clarified allantoic fluid or concentrated cell culture supernatants.
Besides the choice of gradient material, it is important to maintain the integrity of the gradient to not influence the sedimentation behavior of the lysed viral cores and ensure reproducible results. In addition, incomplete resuspension of the pellet fraction after ultracentrifugation likely leads to different outcomes. Finally, we strongly recommend adjusting the pH of the detergent-containing buffer solutions ideally on the same day of the experiment as small changes in ionic concentration might have a drastic effect on the viral core stability.
It is important to note that depending on the particular IAV strain, different disassembly efficiencies might occur based on the properties of the respective M1 and NP variants. This might also apply for mutant viruses. Thus, pH thresholds for matrix layer and vRNP dissociation have to be determined for the respective IAV strain before testing additional influences. For the analysis of specific conditions on viral core stability we suggest adjusting the bottom glycerol layer to a pH value so that half-maximal disassembly of M1 is achieved. It is possible to investigate the influence of specific ions or reducing agents by modifying the detergent-containing layer accordingly. Putative cellular uncoating factors could be added directly to the glycerol gradient. In case of cytoplasmic factors, a third glycerol layer at the bottom of the gradient could be introduced, which is adjusted to neutral pH, contains the cellular factor of interest, and is free of detergent. In this way, the three-layer gradient would even more closely mimic the passage of the virus through the endocytic system and release of gene segments into the neutral cytoplasm.
Taken together, we present a robust yet simple in vitro assay to study IAV priming and uncoating, which has the potential for versatile modifications in order to address diverse questions in the context of IAV entry. In addition, it can be applied to investigate entry processes of other enveloped viruses with related uncoating mechanisms.

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><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.

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 of pH, the bottom layer of the glycerol gradients were adjusted to values between pH 7.4 and 5.0 at a constant salt concentration. X31 derived from clarified allantoic fluid was subjected to velocity gradient centrifugation and analyzed by SDS-PAGE (Figure 2A, B). Without detergent present in the gradient (Figure 2A, lane1) intact virions are pelleted, as reflected by the characteristic pattern of bands representing HA (HA1 and HA2; 63 kDa), NP (56 kDa), and M1 (27 kDa) in the Coomassie stained gel. Since the gel was run under non-reducing conditions, proteolytically cleaved HA1 and HA2 are still connected by disulfide bonds and run at approximately 64 kDa. Under reducing conditions the bands corresponding to HA1 and HA2 would appear very close to the NP and M1 bands, respectively, making it difficult to faithfully interpret and determine band intensities that are solely derived from the viral core (Figure 2B).
Upon addition of NP-40 to the bottom (25%) glycerol layer the viral envelope including HA, NA, and M2 were solubilized and the viral core alone sedimented into the pellet during ultracentrifugation (Figure 1; Figure 2, lane 2). Starting with a pH below 6.5, M1 is gradually lost from the pellet fraction, reaching a minimum between pH 5.8 and 5.4 (Figure 2A, B). Below pH 5.8 vRNPs were dissociated and thus lost from the pellet. Previously, it has been shown that, in fact, whole vRNPs are released into the upper layer, as their release correlated with the loss of PB2 from the pellet fraction16. Protein band intensities were determined, revealing two distinct pH thresholds for disassembly of the M1 layer and dissociation of vRNP bundles, respectively (Figure 2). Additional bands could be observed on the gel, which might correspond to the polymerase proteins PB1 (87 kDa), PB2 (86 kDa), PA (83 kDa), and M2 (11 kDa). Due to their lower copy numbers inside IAV virions we could not reliably determine their identity and they were consequently not taken into consideration for the quantification.
<img alt="Figure 1" src="/files/ftp_upload/53909/53909fig1.jpg" /> 
Figure 1: Schematic representation of the IAV in vitro core disassembly assay. Briefly, purified viral particles are loaded on a two-step glycerol gradient. The bottom layer contains the detergent NP-40 and is adjusted to the desired pH and salt concentration while the upper, detergent-free layer is kept at a constant pH and salt concentration. Depending on the condition in the lower glycerol layer, complete viral cores sediment to the bottom (1) or are dissociated and remain in the glycerol layer (2). Neutral pH leads to sedimentation of intact viral cores composed of M1 and vRNPs. Conditions favorable for uncoating, such as acidic pH (5.0), results in dissociation of the viral core components and therefore loss from the pellet fraction. Following ultracentrifugation, glycerol supernatants are removed and pellets are analyzed by SDS-PAGE. <a href="https://www.jove.com/files/ftp_upload/53909/53909fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53909/53909fig2.jpg" /> 
Figure 2: IAV core disassembly in vitro upon acidification. (A) In vitro disassembly of X31 cores at different pH conditions in the lower glycerol layer. As a control, NP-40 was omitted from the bottom glycerol layer (first lane). Samples were separated by SDS-PAGE under non-reducing conditions followed by Coomassie staining. Viral protein bands are indicated on the right. (B) Densitometric quantification of the intensities of viral protein bands shown in (A). Protein band intensities for M1 and NP were normalized to those at pH 7.4 without NP-40. Data are represented as means of duplicate experiments &#177; standard deviation (SD). Adapted from Stauffer et al., 201416. <a href="https://www.jove.com/files/ftp_upload/53909/53909fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.9K Views.  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.

Video: 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.

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

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.

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.

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.

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.

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

Biology

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.

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.

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.

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

Bioengineering

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.

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 ...

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

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.

Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications

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.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Summary

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Chapters in this video

Preparation of Viral DNA from Nucleocapsids

In vitro Uncoating of HIV-1 Cores

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

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

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

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

Assembly and Purification of Prototype Foamy Virus Intasomes

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

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

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