Merkel Cell Polyomavirus Infection and Detection

Human neonatal foreskins were obtained from Penn Skin Disease Research Center. Adult human fibroblasts were obtained from discarded normal skin after surgery. All the protocols were approved by the University of Pennsylvania Institutional Review Board.

1. Isolation of human dermal fibroblasts

1. Use a pair of scissors to trim off fat and subcutaneous tissue from the human neonatal foreskin and cut the skin sample in halves or quarters.
2. Incubate the tissue in 5 mL of 10 mg/mL dispase II in PBS supplemented with antibiotic-antimycotic at 4 °C overnight.
3. In a sterile 10 cm dish, carefully separate the epidermis from the dermal layers using microdissection forceps.
4. Transfer the resulting dermal tissue to a 15 mL conical tube containing 5 mL of 2 mg/mL collagenase type IV in FBS-free medium supplemented with antibiotic-antimycotic.
5. Incubate the sample at 37 °C in 5% CO₂ for 4-6 h with periodic shaking until just a few macroscopic tissue aggregates remain.
6. Release single cells from the dermis by pipetting up and down (triturating) vigorously ten times with a 10 mL pipette.
7. Centrifuge at 180 x g for 5 min, and discard the supernatant.
8. Plate the dissociated cells in DMEM medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, and 1% L-glutamine at 37 °C in 5% CO₂.

2. 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 2).
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 2.2, 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 293TT²⁸ 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 2.4, 8.4 µg of ST expression plasmid pMtB and 9.6 µg of LT expression plasmid pADL*²⁹.
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 rabbit³⁰) to determine transfection efficiency. At this stage, nuclear LT signal 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 2.8. Most of the cells should be both MCPyV LT and VP1 positive at this stage.
10. To harvest the virus, trypsinize 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 2.11.
13. Combine the supernatants from the step 2.11 and 2.12, and spin one more time as described in step 2.11.
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 rabbit³⁰). Pool gradient fractions with peak dsDNA and/or VP1 content and characterize the stock by quantitative PCR to calculate the viral genome equivalent³¹. Store MCPyV virion stock at -80 °C.

3. Infection

1. Maintain primary human dermal fibroblasts in DMEM with 10% fetal calf serum, 1% non-essential amino acids and 1% L-glutamine. Upon reaching confluence, split fibroblasts 1:4 without spinning down.
   NOTE: For highest MCPyV infection efficiency, use primary fibroblasts between passages 5 and 12 that are actively dividing at the time of plating.
2. To infect human dermal fibroblasts, aspirate the medium and wash the cells with DPBS.
3. Add 1 mL of 0.05% Trypsin-EDTA to the dish and incubate at 37 °C for 5-10 min.
4. Check under the microscope to make sure that the cells are coming off the dish.
5. Add 10 mL of DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF and 3 µM CHIR99021, all of which stimulate MCPyV infection²⁴, to the dish and transfer the cell solution into a 15 mL tube.
6. Spin down the cells at 180 x g for 2 min, and discard the supernatant. Resuspend the cells in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL bFGF and 3µM CHIR99021 at 2-4 x 10⁴ cells per mL.
7. Seed 200 µL of the cell suspension supplemented with 1 mg/mL collagenase type IV into each well of a 96-well plate. Thaw MCPyV virion stock on ice. Add 10⁹ viral genome equivalents of MCPyV virions (calculated in step 2.18) per 1 µL of iodixanol for each 2,500 to 5000 cells to be infected.  Tap the side of the plate gently and place the plate in the incubator.
8. After incubation at 37 °C in 5% CO₂ for 48-72 h, add 20% FBS to each well.
9. Allow the infection to proceed at 37 °C in 5% CO₂ for 72-96 h.

4. Immunofluorescent staining

1. Fix cells obtained in step 3.9 with 3% paraformaldehyde in PBS for 20 min.
2. Wash the cells with PBS twice.
3. Incubate the cells in blocking/permeabilization buffer (0.5% Triton X-100, 3% BSA in PBS filtered through 0.22 µm filter) at RT for 1 h.
4. Incubate the cells with the following primary antibodies: mouse monoclonal anti-MCPyV LT (CM2B4) (1:1000) and rabbit polyclonal anti-MCPyV VP1 antibody (1:2000), at RT for 3 h.
5. Wash the cells with PBS three times.
6. Incubate the cells with the secondary antibodies: Fluor 594 goat anti-mouse IgG (1:1000) and Fluor 488 goat anti-rabbit IgG (1:500) at RT for 1 h.
7. Wash cells with PBS three times.
8. Counterstain the cells with 0.5 µg/mL DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride).
9. Mount coverslips on glass slides and analyze using an inverted fluorescence microscope.

5. In situ DNA-HCR

NOTE: This technique requires that cells be seeded on coverslips. For this purpose, the infection conditions described above (step 3) may be scaled up to the 24-well plate format.

1. Fix cells cultured on coverslips with 4% PFA in PBS for 10 min. Wash the coverslips twice with PBS, then treat them with 70% ethanol to permeabilize the cells at 4 °C overnight.
   NOTE: Fixed samples can remain in this state for several days.
2. Pre-hybridize samples by replacing the ethanol with probe hybridization buffer and incubating for 60 min at RT.
3. Dilute the probe(s) (1 µM) (Table 1) in probe hybridization buffer solution at 1:500 dilution 30 min prior to the end of the pre-hybridization step and incubate at 45 °C.
4. At the end of the incubations, pipette ~10 μL of the diluted probe mix per coverslip on microscope slides. Place the coverslips, cell-side down, on their respective droplets of hybridization probe mix. Seal the edges and backs of the coverslips to the slides with a liberal amount of rubber cement.
5. Heat the slides with added probes at 94 °C for 3 min by setting the slides on the flat side of a heat block. Transfer the slides to a humidified chamber and incubate at 45 °C overnight. To make a simple humidified chamber, lightly dampen sterile paper towels with dH₂O and place in the bottom of a plastic container that seals with a rubber gasket.
   NOTE: During heating the rubber cement can bubble briefly. However, ensure that the seal does not break.
6. Carefully peel away the rubber cement with forceps and place the coverslips cell-side up back into wells of a 24-well plate. Wash the coverslips with probe wash buffer at RT three times.
7. Incubate the coverslip in the amplification buffer (200 µL in 24-well plates) at RT 30-60 min.
8. Meanwhile, anneal each of the two labeled oligonucleotide hairpins recognizing the probes (Table 1) in separate PCR tubes by heating to 94 °C for 90 s and cooling to RT for 30 min while protecting from light. Mix the two hairpins in amplification buffer, each at a dilution of 1:50.
9. Make a surface for the amplification reaction by stretching paraffin film over the open face of a 24-well plate lid. Pipette 50-100 µL droplets of the hairpin/amplification buffer mixture onto the paraffin film for each coverslip. Use forceps to carefully remove the coverslips from the pre-amplification solution. Dry the coverslip by touching the edge to a porous disposable wipe, and place cell-side down onto the amplification droplet.
10. Place the plate lid holding the coverslips into a humidified chamber and incubate overnight at RT in the dark.
11. Return the coverslips to wells once more. Incubate the samples with 5x SSCT [5x sodium chloride sodium citrate (SSC) 0.1% Tween 20 filtered through a 0.22 µm filter] containing 0.5 µg/mL DAPI for 1 h at RT. Wash the samples twice with 5x SSCT at RT.
12. Mount the coverslips on microscope slides. Analyze the cells and image the samples using an inverted fluorescence microscope.