The overall aim of this protocol is to measure BK-polyomavirus non-coding control region driven transcriptional activity by measuring dual fluorescent cells via flow cytometry. The BK-polyomavirus can cause severe pathologies in immunocompromised patients, especially in renal transplant recipients. However, since highly effective antivirus are currently not available, methods measuring the potential anti-viral impact of other compounds are required.
This method allows virologists to analyze the impact of potential anti-viral agents affecting the BK-polyomavirus transcriptional activity driven by the non-coding control region. This assay further allows to study the influence of rearrangements on the promoter activity in comparison to archetypical non-coding control regions. The advantage of studying the viral promoter activity with a dual fluorescence report is that early and late gene expression can be analyzed in a mutually independent manner.
Furthermore, the difficult perforation of virus stocks and long-term infectious cell culture experiments are not necessary since only transfected and fluorescent cells are analyzed, also drugs reducing the cell viability and perforation can be studied. This protocol follows the guidelines of human research as approved by the ethic committee of the medical faculty of the University of Duisburg-Essen. The first step is to collect blood samples for the isolation of polyomavirus DNA.
Collect at least three milliliters of blood in EDTA acquisition tubes. Centrifuge the sample at 2, 500 G for 15 minutes and pipette the plasma into a new tube. Prepare 40 microliters of protein SK into a 1.5 milliliter micro-centrifuge tube and add 400 microliters plasma by pipetting.
Isolate the DNA using a DNA blot extraction kit as described in the manufacturer's instructions. Prepare the master mix for the pre-PCR using primer pair A in a total volume of 50 microliters and use the previously isolated DNA. Distribute 45 microliters of the master mix into the PCR tubes.
Add five microliters of the isolated DNA into the PCR tubes and run the PCR with the reaction conditions as illustrated in table three. Repeat denaturation annealing and extension in 35 cycles. For nested application, use primer pair B, harboring the restriction sites for cloning.
Mix 10 microliters of the PCR product with two microliters of six fold gel loading dye. Load 10 microliters of the mix on a 1.5%agarose gel and run the gel for 30 minutes at 60 milliampere. Visualize the gel using an appropriate UV documentation system.
Purify the PCR amplicons using a PCR purification kit according to the manufacturer's instructions. Digest the purified amplicons with the indicator restriction enzymes for two hours at 37 degrees Celsius. Repeat the purification steps to purify the digested amplicons.
In parallel, also digest the plasmid backbone. Analyze the digested plasmid backbone on a 0.8%low mount agarose gel. Do not run the gel at a higher current than 40 milliampere.
Visualize DNA fragments using long wave UV light and cut out the backbone bent using a clean scalpel. Avoid long UV exposure times to prevent DNA damage. Fence for the piece of flomel gel fragment into a new 1.5 milliliter micro-centrifuge tube.
Heat the backbone containing low mode agarose piece for 10 minutes at 65 degrees Celsius in order to melt the gel piece and mix every two minutes by gentle vortexing. The melted gel can be directly used for ligation. Ligate backbone and the digested amplicon using T4 DNA ligase overnight at 16 degrees Celsius.
After standard cloning procedure, isolate the plasma DNA. Perform restriction enzyme digestion to check for positive clones and visualize digested fragments on a 1%agarose gel. Seed 100, 000 HEK293T cells per well of the 12 well plate 24 hours prior to transfection and incubate overnight at 37 degrees Celsius, maintain active proliferation during transfection.
Cells should be approximately at 80%confluency at transfection. Place 250 microliters of reduced serum media in sterile tube and add one microgram of each reported plasma DNA and mix gently by pipetting. Add three microliters of the transfection reagent to the DNA mixture and mix gently by pipetting and incubate for 15 minutes at 22 degrees Celsius.
Add the mixture, drop-wise to the well and gently distribute to the well. After four hours, replace the supernatant with fresh medium containing the testing agents and solvent control. In this example, the mTOR inhibitors INK-128 and rapamycin was used.
Incubate at 37 degrees Celsius until analysis. Check cells for red and green fluorescence under the fluorescence microscope. Red and green fluorescence correspond to the early and late BK-polyomavirus gene expression respectively.
72 hours post transfection, carefully aspire the supernatant. Wash the cells twice with one milliliter of cold PBS. Gently add 500 microliters trypsin and turn the plate slightly to avoid premature detachment of the cells.
This step is important to avoid cell doublets. After addition, trypsin is removed directly with the same pipette tip. Incubate the cells for at least five minutes at 37 degrees Celsius.
Re-suspend trypsin S cells with one milliliter PBS containing 3%FCS and transfer the suspension in pre-labeled FACS tubes. Add DAPI at a concentration of one microgram per milliliter prior to FACS analysis. Analyze the cells using a flow cytometer.
For each sample, measure at least 10, 000 living cells. Initial compensation is essential to distinguish red and green signals to achieve accurate results. The non-coding control regent was amplified using a nested PCR protocol using the inner primer pair which harbors the restriction sites for cloning.
The amplicon verification was performed via agarose gel electrophoresis While the amplicons derived from archetypical NCCR sequences at a homogenous size distribution in the gel, those derived from rearranged NCCRs with insertions and deletions, differed in their size. Selection of positive clones containing the NCCR was verified by restriction analysis. The small spacer region was replaced by the larger NCCR fragments indicating positive clones.
Plasmid DNA isolated from verified clones were sent for pyrosequencing. In this example, the deletion in the P-block and the substitution in O-block were detected. HEK293T cells were transiently transfected with the respect for reportable construct and NCCR activity was monitored by a fluorescence microscopy.
Red and green fluorescents corresponded to early and late BK-polyomavirus gene expression respectively. The first NCCR displayed a strong late gene expression while the second example had a comparatively strong early but moderate late gene expression. DAPI negative cells were gated and a dot plot was created showing eGFP versus tdTomato.
Samples that were negative as well as those positive for tdTomato or eGFP only were included into the analysis. Mean fluorescence intensities were derived from each testing clone. In this example, the treatment with the dual mTOR inhibitor INK128 significantly reduced tdTomato MFI which means that early expression was inhibited.
This method allows to identify currently used and other compounds to detect the BK-polyomavirus through transcriptional activity. In early gene expression, this decidedly determines the viral implication. Inhibitory drops might be considered to be used as agents for the immunocompromised patients in the case of the BK-polyomavirus reactivation.
