We presented the first cell culture model for Merkel Cell Polyomavirus infection. The protocol includes isolation of the dermal fibroblasts, preparation of the MCPyV virus infection, immunofluorescent staining, and the fluorescent in situ hybridization. This protocol makes it possible to study the MCPyV infectious cycle and interactions with host cells while avoiding artifacts associated with over-expression of viral genes.
To begin, use a pair of scissors to trim the fat and subcutaneous tissue off of the human neonatal foreskin. Cut the skin sample in halves or quarters. Place the tissue in 5 milliliters of 10 milligram per milliliter dis-based II NPBS, supplemented with antibiotic-antimycotic.
Incubate at 4 degrees Celsius overnight. The next day, use microdissection forceps to carefully separate the epidermis from the dermal layers in a 10 centimeter dish. Transfer the resulting dermal tissue to a 15 milliliter conical tube containing 5 milliliters of 2 milligrams per milliliter collagenase type IV in FBS-free medium supplemented with antibiotic-anitmycotic.
Incubate the sample at 37 degrees Celsius in 5%carbon dioxide with periodic shaking for four to six hours, until just a few macroscopic tissue aggregates remain. After this, use a 10 milliliter pipette to pipette the sample solution up and down vigorously 10 times, releasing single cells from the dermis. Centrifuge at 180 g for five minutes and discard the supernatant.
Plate the dissociated cells in supplemented DMEM medium. In the late afternoon or evening, seed 6 million 293TT28 cells into a 10 centimeter dish containing supplemented DMEM medium. Incubate at 37 degrees Celsius with 5%carbon dioxide.
The next morning, ensure that the cells are about 50%confluent, then transfect the cells with 66 microliters of transfection reagent, 12 micrograms of re-ligated MCPyV isolate R17B DNA, 8.4 micrograms of ST expression plasmid pMtB, and 9.6 micrograms of LT expression plasmid pADL. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide. The following day, when the transfected cells are nearly confluent, trypsinize the cells and transfer them to a 15 centimeter dish for continued expansion.
When that dish becomes nearly confluent, transfer the cells into three new 15 centimeter dishes. When those new dishes become nearly confluent, harvest the cells as outlined in the text protocol. Centrifuge at 180 g at room temperature for five minutes.
Then remove the supernatant and add one cell volume of DPBS magnesium. Add 25 micromolar ammonium sulfate, followed by 0.5%Triton X-100, 0.1%benzonase, and 0.1%of an ATP-dependent DNAse. Mix well and incubate at 37 degrees Celsius overnight.
The next day, cool the mixture on ice for 15 minutes. Next, add 0.17 volume of sodium chloride. Mix and incubate on ice for another 15 minutes.
Centrifuge at 12 thousand g at 4 degrees Celsius for 10 minutes. If the supernatant is not clear, gently invert the tube and repeat the centrifugation. After this, transfer the supernatant to a new tube.
Resuspend the pellet in 1 volume of DPBS supplemented with 0.8 moles of sodium chloride. Centrifuge at 12 thousand g at four degrees Celsius for 10 minutes. If the supernatant is not clear, gently invert the tube and repeat the centrifugation.
Combine the two supernatants and centrifuge again at 12 thousand g at four degrees Celsius for 10 minutes. Then, use a pipette to deposit gradients of iodixanol into thin-walled five milliliter polyallomer tubes as outlined in the text protocol. Load 3 milliliters of the clarified virus-containing supernatant on top of the prepared iodixanol gradient.
Centrifuge with an SW 55 Ti rotor at 234 thousand g and at 16 degrees Celsius for 3.5 hours, making sure to set the acceleration and deceleration to slow. After the ultracentrifugation is complete, collect 12 fractions in siliconized tubes. After maintaining and detaching the cells, at 10 milliliters of supplemented DMEM and F12 medium to the dish.
Transfer the cell solution to a 15 milliliter tube. Centrifuge at 180 g for two minutes. Discard the supernatant and re-suspend the cells in supplemented DMEM and F12 medium at a cell density between 20 thousand and 40 thousand cells per milliliter.
Then, seed 200 or 400 microliters of the cell suspension supplemented with 1 milligram per milliliter of collagenase type IV into each well of a 24 or 96 well plate, respectively. Remove the MCPyV viron stock from the minus 80 degree Celsius freezer and place on ice to thaw. Add 1 billion viral genome equivalents of MCPyV virions per one microliter of iodixanol for each 2500 to 5000 cells to be infected.
Tap the side of the plate gently, and incubate at 37 degrees Celsius with 5%carbon dioxide. First, fix the cells onto cover slips with 4%PFA in PBS for 10 minutes, then wash the cover slips twice with PBS and treat them with 70%ethanol at four degrees Celsius overnight to permeabilize the cells. The next day, replace the ethanol with probe hydration buffer and incubate at room temperature for 60 minutes to pre-hybridize the samples.
30 minutes before the end of the pre-hybridization incubation, dilute the probes in probe hybridization solution at a 1:500 dilution and incubate at 45 degrees Celsius. When the incubations are complete, pipette approximately 10 microliters of the diluted probe mixture onto a microscope slide for each cover slip. Place each cover slip cell side down on its respective droplet of hybridization mix.
Using a liberal amount of rubber cement, seal the edges and back of each cover slip to its slide. Set the slide on the flat side of a heat block and heat them to 94 degrees Celsius for three minutes. After this, transfer the slide to a humidified chamber and incubate them at 45 degrees Celsius overnight.
The next day, use forceps to carefully peel away the rubber cement. Place the cover slips cell side up into the wells of a 24 well plate and wash them with probe wash buffer three times at room temperature. Add 200 microliters of amplification buffer to each well containing a cover slip and incubate at room temperature for 30 to 60 minutes.
Meanwhile, anneal each of the two labeled oligonucleotide hairpins recognizing the probes in separate PCR tubes by heating them to 94 degrees Celsius for 90 seconds, and then cooling them to room temperature for 30 minutes. Mix the hairpins together in amplification buffer at a dilution of 1:50. Next, stretch paraffin film over the open face of a 12 well plate lid to make a surface for the amplification reaction.
Pipette 50-100 microliter droplets of the hairpin mixture onto the paraffin film for each cover slip. Using forceps, carefully remove each cover slip from the pre-amplification solution and touch the edge to a porous disposable wipe to dry. Place each dried cover slip cell side down onto an amplification droplet.
Transfer the plate lid into a humidified chamber and incubate overnight at room temperature and in the dark. The next day, return the cover slips to the wells of the 24 well plate. Add 5X SSCT containing DAPI at a concentration of 0.5 micrograms per milliliter to each well and incubate at room temperature for one hour.
After this, wash the samples twice with 5X SSCT at room temperature. Mount the washed cover slips onto microscope slides, and use an inverted fluorescence microscope to analyze the cells and image the samples. In this study, an nearly-homogenous population of human dermal fibroblasts is isolated.
Immunofluorescent staining reveals that almost 100%of the isolated human dermal cells are positively stained for dermal fibroblast markers, vimentin, and collagen, but negative for human foreskin keratinocyte marker K14. MCPyV virions are then generated and after visualizing the band of MCPyV virions concentrated in the core of the gradient, 500 microliter fractions are collected and MCPyV qPCR is performed to identify the peak fractions. Immunofluorescent stained images of HDFs infected with MCPyV are shown here, where cells that are LT positive, VP1 positive, or both LT and VP1 positive are considered to be positive for MCPyV infection.
Over 30%of cells are LT positive, and more than 10%are VP1 positive. The MCPyV genomes replicated in the infected cells are detected using both the HCR-DNA FISH and Immunofluorescent-HCR-DNA FISH. While the HCR-DNA FISH reveals the localization of MCPyV DNA present in the replication factory, the Immunofluorescent-HCR-DNA FISH methods allow simultaneous detection of both MCPyV DNA and LT protein co-localizing at the replication centers.
To achieve the best virus infection efficiency, it's very important that MCPyV is concentrated to at least 2 x 10 to 8th virus genome per microliter since iodoxanol is toxic to the cells. Following MCPyV infection of the dermal fibroblasts, many molecular biologic analyses can be performed, including immunofluorescence, immuno-blocking, immunopositivication, and qPCR-based assay. The infection system that we've described makes it possible for researchers to study stages of MCPyV infection like nuclear trafficking and viral packaging.
MCPyV is a BSL-2 virus, but little is known about its potential for pathology in the amounts achieved in a laboratory setting. Therefore, researchers should take precautions to avoid exposure to the virus.
