Dye Labeling HPV Capsids

 Jeff Roberts and Chris Buck
John Schiller’s Group
Laboratory of Cellular Oncology, NCI

Laboratory of Cellular Oncology, NCI

Citations:  PMID:  17603495 and Labelin18773072

Purpose:  covalent conjugation of a fluorochrome to HPV capsids.  

Fluorescently labeled capsids can be used for HPV binding and entry studies.  Although fluorescent capsids can also be produced using L2-GFP or –RFP fusion proteins (plasmids puL2f and puL2t), dye-conjugated capsids are brighter, more photo-stable and less pH-sensitive.

Overall, the procedure basically follows the protocol supplied with an Alexa Fluor 488 protein labeling kit, Invitrogen cat #A10235. The fluorochrome is amine-reactive.  Since the chemistry involved requires deprotonation of lysine side-chains, the kit protocol recommends performing the conjugation reaction at alkaline pH.  In our hands, the 100 mM bicarbonate called for in the kit protocol does not significantly harm the infectious titer of pseudovirus stocks.

Small molecules, such as the Alexa Fluor 488 dye, can freely pass through holes in the HPV capsid.  Therefore, the conjugation reaction results in labeling of both L1 and L2, as well as the histones within the capsid lumen.

The kit protocol calls for a total protein concentration of 2 mg/ml.  Although it’s possible to produce purified capsid stocks this concentrated using vector propagation methods, a simpler approach is to conjugate the dye to capsids in the setting of clarified cell lysate, prior to capsid purification.  The labeled capsids can then be purified away from free dye and lysate components using standard Optiprep ultracentrifugationmethods.

Invitrogen refuses to specify how much dye is distributed into the individual tubes in the A10235 labeling kit.  If a bulk stock of dye (e.g. Invitrogen # A20009) is used, 17 µg of dye per labeling reaction would be a good initial guess.

Procedure

•Co-transfect a T-225 flask of 293TT with a packaging plasmid (e.g., p16sheLL) and a red fluorescent protein plasmid (e.g., pRwBor ptwB) according to the Standard Protocol.  Alternatively (preferably), infect cells with a propagatable pseudovirus seed stock.

•Harvest cells 48 hours after transfection or 72 hours after infection, according to main protocols.  DO NOT add ammonium sulfateto the lysate.  The free ammonium may quench the amine-reactive TFP ester on the Alexa 488 fluorochrome.  Either allow the capsids to mature in the unbuffered (acidic) lysate or buffer the lysate by adding 40 mM sodium phosphate from a 1M stock buffered to pH 7.5.

•Expect roughly 600 µl of lysate.  Allow capsids to mature by incubating the lysate at 37ºC for 24 hours.

•Add 0.17 volume of 5 M NaCl, clarify the lysate by centrifuging 10 minutes at 5,000 x g.  Transfer clarified supe to a fresh tube.  Optional: re-clarify the supe 10 minutes at 5,000 x g.

•Determine protein concentration of the clarified lysate using BCA reagent.  Expect roughly 8-12 mg/ml.

•Add water to dilute the lysate to 3-4 mg/ml. 

•Make 1 M bicarb buffer.  A bicarb vial comes with the Invitrogen Alexa Fluor 488 Kit (#A10235).  Alternatively, make 1 M sodium bicarbonate in water from scratch.   Bicarb takes quite a while to go into solution.  

•Add 50 µl of bicarb buffer to 500µl of diluted lysate.  Transfer the mixture into an Alexa Fluor tube while vortexing gently (caution:  vigorous vortexing may flip the stir bar out of the tube).  Transfer the tube to a stir plate for one hour at room temperature, protected from light.

•After the conjugation reaction, neutralize the pH by adding 20 µl of 1M NaPO₄pH 6.5

•Perform Optiprep gradient purification.  The conjugated particles should be visible as a distinct orange-green band within the gradient.

•Optional: pool L1-positive gradient fractions and perform agarose Gel Filtrationto remove Optiprep and further purify the particles. Optiprep isn’t cytotoxic and doesn’t interfere with most types of assays and very high particle purity is generally achievable.  So gel filtration shouldn’t typically be necessary.

•Run selected fractions on a 4-12% NuPage Gel.  Visualize the bands by fluorescent imaging of the unfixed/unstained gel.  Expect coupling of the dye to L1, L2 and histones.  Stain gel with Microwave Blue.  

•Pool L1⁺fractions.  Expect a particle concentration of ~200 ng/µl.

•Check particle binding to HeLa cells  (dislodge cells with 5 mM EDTA in PBS or use CellStripperreagent).  ~10 ng of labeled capsids on 30,000 suspended HeLa cells should give reasonable signal by FACS (signal:noise ratio of at least 3).

Older methods(See “Citations” above)

In initial conjugated capsid preparations, we made use of capsid stocks purified using an Optiprep equilibrium gradient.  Equilibrium gradient stocks contain a substantial amount of cellular protein that serves as a sink for excess dye.  The cellular proteins and free dye can easily be removed using agarose gel filtrationafter the conjugation reaction.  Another successful strategy was to first purify capsids using a standard Optiprep step gradient, then use BSA to bring the total protein concentration up to 2 mg/ml in PBS.  Optiprep and 0.4 M NaCl do not appear to interfere with the conjugation reaction.

Acknowledgement

We are gateful to Jason Smith for sharing the results of his experiments identifying appropriate dye-to-protein ratios.