Lyco negative strand, RNA viruses. The genome of influenza viruses is packaged in the form of viral rib nuclear protein complexes, or VR nps in which the single stranded genome is encapsulated by the nuclear protein and associated with the turmeric polymerase complex consisting of the PA PB one and PB two subunits to purify intact complexes from infected mammalian cells. Hela cells are incubated with recombinant strept tagged influenza virus.
Once the virus enters the cells, the viral RNA is synthesized in the nuclei following infection, the cells are lies to using a detergent and fractionated into cytoplasmic nuclear and chromatin fractions using centrifugation and assault based chromatin fraction protocol. Finally, the strept tagged VRPs are affinity purified from each fraction analysis of the protein and RNA composition of purified VR NPS by silver staining demonstrates that using this protocol even tightly, chromatin bound VRPs can be isolated.Intact. So one of the main advantages of our protocol when compared to standard methods for affinity purifying influenza virus rib nuclear protein complexes is that by using our method, it's possible to purify VR NPS from the chromatin of infected cells, which is known to be the site of influenza virus mRNA synthesis.
So our method could be used to answer questions surrounding the regulation of viral replication and transcription, such as, for instance, which viral or cellular factors, but also which post-translational modifications are required for, for instance, viral mRNA synthesis or also viral genomic RNA synthesis. So researchers new to this method might run into some difficulty because sub nuclear fractionation is very sensitive to biochemical conditions and it can also vary greatly between different cell lines. Begin this protocol with heal our cells that have been infected with influenza virus, RWSN PB two strep, which has a strep tag genetically fused to the C terminus of the PB two subunit at a multiplicity of infection of three.
Wash the cells once with cold PBS, then add 12 milliliters of cold PBS and remove them from the dish. Using a rubber scraper. Transfer the cells to a 50 milliliter conical tube and pellet them by centrifugation.
The most difficult aspect of this procedure is the cell fractionation protocol. Cell nuclei are very sensitive to homogenization and mixing procedures, and if it's performed incorrectly, it can easily ruin a chromatin extract. So it's very important to follow not just the homogenization techniques, but also the just the general handling of lysates as we show here.
Following the spin aspirate, the PBS and Resus, suspend the cell pellet in 10 milliliters of cold sucrose buffer. Attach a 200 microliter pipette tip to a 10 milliliter plastic serum pipette and pass the cells through it to break up cell clumps. Incubate the suspension on ice for 10 minutes.
Periodically inverting the cells gently to resuspend them. Next, add non IET P 40 to a final concentration of 0.5%and vortex at high speed for 15 seconds immediately centrifuge for 10 minutes at 3, 500 times G at four degrees Celsius in a tabletop centrifuge. After the spin, remove the supinate and transfer it to a 15 milliliter to conical tube.
Store the supinate at four degrees Celsius. This is a cytoplasmic fraction. The pellet should be smaller and whiter than the original cell pellet as shown here.
If the protocol will be completed at a later time, store the cell pellet at four degrees Celsius. Otherwise, continue by washing the pellet in five milliliters of cold sucrose, buffer, and centrifuging as before. Once the spin has completed, discard the supinate and resuspend the pellet, which contains the nuclei in 1.5 milliliters of cold nuclear plasmic extraction buffer.
Transfer the resuspended pellet to a chilled all glass four milliliter dance homogenizer with a tight fitting pestle and homogenize the nuclei with 20 strokes. Avoiding foaming, the resistance should increase after approximately 10 strokes. To confirm efficient homogenization, examine the sample under a phase contrast microscope.
The nuclei should no longer be round, but have irregular forms and appear as though they have burst. Once the nuclei have been homogenized, transfer them to a 1.5 milliliter micro centrifuge tube. Incubate the homogenate on a rotating wheel at four degrees Celsius for 20 minutes.
Then centrifuge at 16, 000 times G in a micro centrifuge for 10 minutes at four degrees Celsius. After the spin transfer the supinate, which is the nuclear plasmic fraction to a 1.5 milliliter tube, and store it at four degrees Celsius. Then Reese suspend the pellet in 1.5 milliliters of digestion, buffer prewarm to 37 degrees Celsius.
Then incubate the fraction for 10 minutes at 37 degrees Celsius. Next, add 42 microliters of five molar sodium chloride to bring the final concentration of sodium chloride to 150 millimolar then incubate for 20 minutes on ice. Centrifuge at 16, 000 times, G in a micro centrifuge of four degrees Celsius for 10 minutes.
After spinning, remove the supinate and store it at four degrees Celsius. This is the CH one 50 fraction. Reeses bend the pellet in 1.5 milliliters of cold high salt buffer and incubate for 20 minutes on ice.
Centrifuge at 16, 000 times, G in a micro centrifuge at four degrees Celsius for 10 minutes. After the spin, remove the sup natant and store it at four degrees Celsius. This is the CH 500 fraction.
Take out the fractions saved during the fractionation protocol and place them on ice. Then to the cytoplasmic fraction, add 300 microliters of five molar sodium chloride. Then transfer 100 microliters of packed strep T in Sphero beads per fraction to a 15 milliliter conical tube.
Add 10 volumes of strep wash buffer to the beads. Invert the tube to mix and centrifuge for five minutes at 1000 times. G in a tabletop centrifuge, discard the sna.
Repeat the buffer wash twice, and then Reese bend the pellet in an equal volume of strep wash buffer, add 200 microliters of washed streps Act in bead slurry to each fraction. Rotate the tubes for one hour at four degrees Celsius. Then centrifuge the tubes for one minute at 1000 times G of four degrees Celsius, discard the supinate.
Then add one milliliter of wash buffer to the beads for beads incubated with the cytoplasmic fraction in a 15 milliliter tube. Add the wash buffer and transfer the beads to a 1.5 milliliter tube. Wash the beads for one minute, then centrifuge the tubes for one minute at 1000 times G at four degrees Celsius.
Repeat twice after the last wash step. Remove all traces of the wash buffer using a flat pipette tip. Then add 100 microliters of elucian buffer and gently mix and place the tubes on ice for 15 minutes.
During the incubation, occasionally mix the beads by gently tapping the bottom of the tubes. After 15 minutes have passed centrifuge to choose for one minute at 1000 times G at four degrees Celsius. Collect the Senna containing the eluted strep tagged VRPs.
He, our cells were infected with influenza virus strain, WSN for nine hours, and the fractionated lysates were analyzed by western blossoming as shown here. Gentle nuclear isolation efficiently extracts cytoplasmic proteins such as tubulin while avoiding the release of abundant nuclear proteins. Note that the chromatin fraction extracted with low salt is enriched in proteins important for transcription.
While the high salt fraction contains primarily nucleosomes to determine the protein composition of the eluted cytoplasmic fraction, V NPS from he R cells infected for nine hours with untagged virus, RWSN or RWSN PB two strep were analyzed by silver staining as shown for a cytoplasmic eluate. Here there is a high ratio of NP to PA PB one, PB two as evidenced by the major band above 55 kilodaltons compared to the bands between 95 and 100 kilodaltons suggesting that most strep PB two is purified as part of a fully formed VRNP, or in other words, little soluble polymerase is captured to compare relative yields. VR NPS purified following nine hours of infection with RWSN PB two strep or RWSN or silver stained as shown in this silver stain gel v RMPs can be purified from all four cellular fractions after nine hours of infection.
The highest concentration of v RMPs is found in the nuclear plasm, although sizable portions can also be purified from the cytoplasm and chromatin. After watching this video, you should have a good understanding of how to purify high yields of intact influenza virus ribonucleoprotein complexes from the nucleus of infected cells. When carrying out this procedure, it's important to remember to use clean buffers and to avoid exposing the samples unnecessarily to room temperature because that way you can avoid both RNA and protein degradation, as well as avoid protein complex disassembly.
And don't forget that working with an infectious influenza virus can be hazardous. All steps should be carried out under a biosafety level two workbench until NP 40 has been added to the lysates.
