In Vitro Disassembly of the Influenza A Virus Core Using pH-Controlled Gradient Centrifugation

الملخص

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

This video demonstrates the pH-dependent disassembly of influenza A virus (IAV) cores using gradient ultracentrifugation. It outlines glycerol gradient preparation, ultracentrifugation, and how different pH conditions affect IAV core disassembly.

البروتوكول

1. Preparation of Buffers and Stock Solutions

1. Prepare MNT buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (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.
2. 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.
3. 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.
4. 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 µm pore size) into a glass bottle.
5. Prepare 10% NP-40 solution and 1 M NaCl in dH₂O.
6. Prepare 25x concentrated protease inhibitor by dissolving two tablets in 4 ml ddH₂O.

2. Preparation of Glycerol Gradients

1. 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.
2. For each pH condition pipet 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 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 HCI or 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.

3. 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 a 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 corresponding to around 20-30 µg of total viral protein, for each gradient.
4. Balance opposing tubes and place them into a SW41 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.

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
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
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
NP-40	Sigma-Aldrich	I8896	Now commercially available as IGEPAL® CA-630
pH indicator strips, pH 4.0-7.0	Merck Millipore	109542
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