Skip to end of metadata
Go to start of metadata

Harvesting BK Polyomavirus (BKV) and JCV Virions

 Laboratory of Cellular Oncology, NCI

 

Citation: Pastrana et al. (2012) “Neutralization serotyping of BK polyomavirus infection in kidney transplant recipients” PLoS Pathogens 8:e1002650

 

Purpose: pseudovirus/reporter vector production methods originally developed for HPVs generally work well for polyomaviruses such as Merkel cell polyomavirus and murine polyomavirus.  Unfortunately, some preparations of BK polyomavirus (BKV or BKPyV) reporter vectors fail to effectively penetrate Optiprep gradients. This is hypothetically due to the ability of the BKV capsid to attract a “shroud” of glycolipids during the cell lysis process.  The problem has historically been dealt with by treating harvested virions with neuraminidase prior to ultracentrifugation (PMID:173877, note: “receptor destroying enzyme” = neuraminidase) and PMID: 6304724). Treatment with neuraminidase removes sialic acids from the headgroups of the glycolipids that BKV capsids bind in a sialic acid-dependent manner.  Hypothetically, the release of the glycolipids from the surface of the BKV capsid increases the apparent density of the capsid, allowing it to effectively migrate down to the denser core of the Optiprep gradient.

 

Protocol Modifications:

 

•Perform Standard Production protocol for generation of VLPs or Revised Production protocol for generation of transduction-competent reporter vectors. 

 

•Trypsinize the cells and wash once with DPBS.  Measure the volume of the cell pellet then suspend the pellet in an equal volumeof DPBS.  The main protocol says to suspend in 1.5 pellet volumes of DPBS. However, addition of neuraminidase stock (see below) takes up additional volume that would result in the lysate being too dilute.  Lysates that are too dilute result in the appearance of background proteins migrating down the Optiprep gradient.

 

•Note: there seems to be a frustrating degree of variability in BKV titer yields when different members of the lab harvest BKV reporter vectors using the Revised Production protocol.  We suspect that some subtle protocol variable is involved. 

 

•Dissolve a 10U vial of Neuraminidase V (Sigma N2876) in 400ul of sterile filtered (10mM phosphate pH 6, 100mM KCl, 0.1% BSA).  Resulting NA stock is 25 U/ml.  Store at 4ºC.  Stock seems reasonably effective for at least a year.

 

•Make 1M Tris pH 8.0. Sterile filter

 

•Make a 10% solution of Brij-58 detergent in water. We have the sense that using Brij-58 instead of Triton X-100 may help combat the Optiprep buoyancy problem, particularly for JCV. Unfortunately, 10% Brij-58 stock solution is only stable for about a week. Brij-58 stock solution should thus be made up fresh within a few days prior to use. Note that it takes quite awhile for Brij-58 paste to go into solution.

 

•Suspend cells at >100 million cells / ml in DPBS.

 

•Add neuraminidase to a final concentration of 2 U/ml.  Incubate 15 minutes at 37ºC, with the goal of chewing away cell surface sialic acids.  We suspect that chewing away the sialic acid prior to detergent lysis of the cell is helpful.

 

•Add Tris (starting from 1M pH 8 stock) to a final concentration of 50 mM. We suspect that exposure of the virus to acidity might be at play in the buoyancy problem. If so, it is important to buffer the cell suspension before adding detergent.

 

•Lyse the cells by adding Brij-58 to a final concentration of 0.5%. 

 

• If harvesting VLPs, add Benzonase.  If infectious titer is the main goal, add RNase A/T1 mix (see Revised Production).

Last updated by Buck, Christopher (NIH/NCI) [E] on Oct 21, 2018