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.
