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

Take centrifugation tubes containing detergent-free glycerol.
For the control sample, add high-concentration, detergent-free glycerol at neutral pH.
For test samples, add high-concentration glycerol containing detergent, adjusted to different pH values.
Overlay the influenza A virus (IAV), containing a lipid envelope and a core composed of matrix protein and ribonucleoproteins.
Perform ultracentrifugation.
In the control, viruses pass through the detergent-free gradient and sediment as intact virions.
In the test samples, detergent in the lower layer solubilizes the viral envelope without disrupting the core.
At neutral pH, the intact core remains stable and sediments.
At mildly acidic pH, which mimics early endosomal conditions, the matrix protein dissociates, leaving the ribonucleoproteins to sediment.
At a more acidic pH, which mimics late endosomal conditions, the viral core fully disassembles, leaving no components to sediment.
This enables in vitro analysis of the IAV core disassembly under different pH conditions.

Using a five-milliliter syringe and a needle, add three milliliters of 15% glycerol solution to six ultraclear centrifugation tubes. Wash the needle in doubly-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 21,000 rpm and 12 degrees Celsius for 150 minutes.