Visualizing the Effect of pH on Solubilization of the Influenza A Viral Core

초록

Source: Stauffer, S. et al., In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation. J. Vis. Exp. (2016)

This video demonstrates the effect of pH on the solubilization of influenza A viral cores. Gel electrophoresis and protein staining reveal the progressive disassembly of the core structure under acidic conditions.

프로토콜

1. Preparation of Glycerol Gradients

1. Prepare 5 ml detergent buffer master mix (300 mM sodium chloride or NaCl, 2% non-ionic detergent (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.
2. For each pH condition, pipette 4.4 ml of the master mix into a 50 ml conical tube.
3. 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 2-(N-morpholino) ethanesulfonic acid (MES) stock solution.
4. 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.
5. 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 hydrochloric acid (HCI) or sodium hydroxide (NaOH) solutions, respectively.
6. 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.
7. Prepare 15% glycerol solution by mixing 50% glycerol stock with dH₂O in a 3:7 ratio.

2. Ultracentrifugation of influenza A virus (IAV)

1. 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 on 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.
2. 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 not to 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.
3. Gently overlay the glycerol gradients with 30 µl clarified allantoic fluid containing IAV (X31, H3N2) diluted in 1 ml MNT buffer (corresponds to around 20-30 µg of total viral protein) for each gradient.
4. Balance opposing tubes and place them into a SW41 swing ultracentrifugation rotor. Centrifuge for 150 min, at 55,000 x g, and 12 °C.
5. 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 µl (1x) non-reducing lithium dodecyl sulfate (LDS) sample buffer. It is important to pipette up and down several times to dissolve the pellet completely. Transfer the sample into a 1.5 ml microcentrifuge tube.

3. SDS-PAGE of Pellet Fractions and Coomassie Staining

1. Heat all samples at 95 °C for 10 min. At this point, the samples could be stored at -20 °C until they are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
2. Load 20 µl 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.
3. Make fixation solution with 40% methanol and 10% glacial acetic acid in ddH₂O.
4. 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.
5. 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 °C until it is scanned for band quantification.
6. 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).

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