Recombinant Merkel Cell Polyomavirus (MCPyV) Virion Preparation: A Technique to Produce High-titer Recombinant MCPyV Virion

1. Recombinant MCPyV virion preparation

1. Digest 50 µg of pR17b plasmid (carrying MCPyV genome) with 250 U of BamHI-HF in a 200 µL volume (4 h at 37 °C) to separate the viral genome from the vector backbone (Figure 1).
2. Add 1200 µL of buffer PB (supplemented with 10 µL of 3 M NaAc, pH 5.2) to the digested DNA and purify over 2 miniprep spin columns (20 µg DNA capacity). Elute the digested pR17b plasmid from each column with 200 µL of TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA).
3. Prepare the ligation reaction in a 50 mL centrifuge tube. Add 400 µL of purified plasmid DNA from step 1.2, 8.6 mL of 1.05x T4 ligase buffer and 6 µL of high concentration T4 ligase. Incubate at 16 °C overnight.
4. Add 45 mL of buffer PB (supplemented with 10 µL of 3 M NaAc, pH 5.2) to the ligation and use a vacuum manifold to load through 2 miniprep spin columns. Elute each column with 50 µL of TE buffer. Expect a yield of about 30 µg of DNA.
5. In the late afternoon/evening, seed 6 x 10⁶ 293TT cells into a 10 cm dish containing DMEM medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, and 1% L-glutamine without hygromycin B.
6. The next morning, ensure that the cells are about 50% confluent. Transfect using 66 µL of transfection reagent (1.1 µL/cm²), 12 µg of re-ligated MCPyV isolate R17b DNA from step 1.4, 8.4 µg of ST expression plasmid pMtB and 9.6 µg of LT expression plasmid pADL.
7. When the transfected cells are nearly confluent, the following day, trypsinize the cells and transfer them to a 15 cm dish for continued expansion.
8. Optionally take a small number of 293TT cells upon expansion and perform IF staining for MCPyV LT (CM2B4) and VP1 (MCV VP1 rabbit) to determine transfection efficiency. At this stage, nuclear LT signals may be visible, but VP1 expression probably will not be detectable.
9. When the 15 cm dish becomes nearly confluent (usually 5-6 days after initial transfection), transfer the cells into three 15 cm dishes. Harvest the cells from the 15 cm dishes when they become nearly confluent and follow the virus harvest protocol below.
   NOTE: Optionally perform quality control IF as described in step 1.8. Most of the cells should be both MCPyV LT and VP1 positive at this stage.
10. To harvest the virus, trypsinize the cells, spin at 180 x g for 5 min at RT and remove the supernatant. Add one cell pellet volume of DPBS-Mg (DPBS with 9.5 mM MgCl₂ and 1x antibiotic-antimycotic). Then, add 25 mM ammonium sulfate (from a 1 M pH 9 stock solution) followed by 0.5% Triton X-100 (from a 10% stock solution), 0.1% Benzonase, and 0.1% of an ATP-Dependent DNase. Mix well and incubate at 37 °C overnight.
11. Incubate the mixture for 15 min on ice and then add 0.17 volume of 5M NaCl. Mix and incubate on ice for another 15 min. Spin for 10 min at 12,000 x g in a 4 °C centrifuge. If the supernatant is not clear, gently invert the tube and repeat the spinning step. Transfer the supernatant to a new tube.
12. Resuspend the pellet using one volume of DPBS supplemented with 0.8 M NaCl, and spin again, as described in step 1.11.
13. Combine the supernatants from step 1.11 and 1.12, and spin one more time as described in step 1.11.
14. Pour gradients of iodixanol in thin wall 5 mL polyallomer tubes by underlaying (27%, then 33%, then 39%) ~0.7 mL steps using a 3 mL syringe fitted with a long needle or a p1000 pipette.
15. Load 3 mL of clarified virus-containing supernatant on the prepared iodixanol gradient.
16. Spin for 3.5 h at 234,000 x g and 16 °C in an SW55ti rotor. Set the acceleration and deceleration to slow.
17. After ultracentrifugation, collect 12 fractions in siliconized tubes (each fraction is ~400 µL).
18. Analyze the fractions for the presence of virus by dsDNA reagent and/or Western blot for VP1 (MCV VP1 rabbit). Pool gradient fractions with peak dsDNA and/or VP1 content and characterize the stock by quantitative PCR to calculate the viral genome equivalent. Store MCPyV virion stock at -80 °C.