Virus-like particles (VLPs)

West Nile Virus VLP protocol

The stable 293T cell line has been transfected with a pVRC8400 expression vector containing structural cassette with the prM-E sequence from the genotype NY99 sequence (provided by Angelica Medina-Selby, Doris Coit and Colin McCoin) preceded by the tissue plasminogen activator signal sequence.

One can typically plan on getting a sample of VLPs within 10 days. Schedule [day of prep indicated]

1. [day 0] Thaw 1 tube of cells per T150 flask, expand in DMEM with 10% FBS and Pen/Strep (supplemented with 1ug/mL puromycin) to desired number of flasks (typically seed one 1700 cm² roller bottle (Corning product #: 431135) with one T150 flask). Takes 1-2 days to expand the cells. All mammalian cells grown at 5% CO2, 37°C.

2. [day 2] Seed rollerbottle with confluent T150 flask, incubate for 3-4 days until rollerbottle is confluent. (Typically 6 roller bottles will provide sufficient material for several experiments).

3. [day 5] Replace DMEM with Gibco FreeStyle 293 medium (Life Technologies) serum-free medium (175 mL per roller bottle).

4. [day 6] Harvest Freestyle 293 medium containing VLPs the next day, spin at low speed (2000 rpm) to clear cell debris. Replace with an additional 175 mL Freestyle medium for second harvest.

5. [day 7] Add PEG 8000 (0.8 g/10 mL medium), and NaCl (0.23 g/10 mL). Nutate at 4°C.

6. [day 8] Harvest again, Add PEG 8000 and NaCl, Nutate overnight at 4°C.

7. [day 9] Spin precipitated medium in JA 14 (mid-speed rotor) for 5 minutes at 10000 rpm. Decant the supernatant, leaving ~2 mL in bottle. Gently rock and re-suspend precipitated VLPs (should be visible as white film on the side of the bottle).

8. [day 9] Spin in table top centrifuge to pellet VLPs (15 min. 3800 rpm).

9. [day 9] Re-suspend VLPs in buffer (20mM Tricine, pH 7.8, 140mL NaCl, 0.005% Pluronic F-127). Nutate overnight at 4°C to re-suspend.

10. [day 10] Prepare Optiprep velocity gradient 55%-45%-35%-30%-25%-20%-10% steps. Spin in a SW41 rotor 34000 rpm, 4°C for 2 hr. 20 min.. Collect band between 35 and 30% steps. Bands above also have VLPs containing the E protein, but are not of homogeneous size and shape (as evaluated negative stain EM). VLPs can be stored in the optiprep containing fractions for 1-2 weeks at 4°C. (I have not tested the stability of the material after freezing at -80°C.)

11. On day of experiment, label VLPs with DiD at ~20-fold molar excess to the protein (E) concentration. Remove excess dye with NAP-10 desalting column. I typically do not use labeled VLPs after 2 days.

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