Name: measurement of bk-polyomavirus non-coding control region driven transcriptional activity via flow cytometry
Uploaded: 2019-07-13T14:00:00
Description: Watch this Scientific Journal Video about Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry at JoVE.com

The authors thank Barbara Bleekmann for excellent technical assistance. These studies were supported by the IFORES-program of the University of Duisburg-Essen Medical School and the RIMUR-program of the University Alliance Ruhr and Mercator Research Center Ruhr (MERCUR). The authors thank the J&#252;rgen-Manchot-Stiftung for the doctoral fellowship of Helene Sertznig and constant support. The collection and use of patient material has been approved by the ethics committee of the medical faculty of the University Duisburg-Essen (14-6028-BO).

This protocol follows the guidelines of human research as approved by the ethic committee of the medical faculty of the University of Duisburg-Essen (14-6028-BO).
1. Collection of blood or urine samples and isolation of polyomavirus DNA
<ol>
	<li>&#160;Collect at least 3 mL of blood in EDTA tubes or urine in acquisition tubes.</li>
	<li>Centrifuge the sample at 2,500 x g for 15 min. If needed, pipette plasma into a new tube and store the plasma samples at 4 &#176;C for several days ...

<ol>
	<li>Drachenberg, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+patterns+of+polyomavirus+nephropathy:+correlation+with+graft+outcome+and+viral+load.">Histological patterns of polyomavirus nephropathy: correlation with graft outcome and viral load.</a> American Journal of Transplant. 4 (12), 2082-2092 (2004).</li><li>Korth, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+low-level+BK+polyomavirus+viremia+on+intermediate-term+renal+allograft+function.">Impact of low-level BK polyomavirus viremia on intermediate-term renal allograft function.</a> Transplant Infectious Disease. 20 (1), (2018).</li><li>Hirsch, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+perspective+on+human+polyomavirus+infection,+replication+and+disease+in+solid+organ+transplantation.">European perspective on human polyomavirus infection, replication and disease in solid organ transplantation.</a> Clinical Microbiology and Infection. 20 Suppl 7, 74-88 (2014).</li><li>Masutani, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Banff+2009+Working+Proposal+for+polyomavirus+nephropathy:+a+critical+evaluation+of+its+utility+as+a+determinant+of+clinical+outcome.">The Banff 2009 Working Proposal for polyomavirus nephropathy: a critical evaluation of its utility as a determinant of clinical outcome.</a> American Journal of Transplantation. 12 (4), 907-918 (2012).</li><li>Korth, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+immune+suppressive+agents+on+the+BK-Polyomavirus+non+coding+control+region.">Impact of immune suppressive agents on the BK-Polyomavirus non coding control region.</a> Antiviral Research. 159, 68-76 (2018).</li><li>Hirsch, H. H., Yakhontova, K., Lu, M., Manzetti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BK+Polyomavirus+Replication+in+Renal+Tubular+Epithelial+Cells+Is+Inhibited+by+Sirolimus,+but+Activated+by+Tacrolimus+Through+a+Pathway+Involving+FKBP-12.">BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP-12.</a> Amerian Journal of Transplantation. 16 (3), 821-832 (2016).</li><li>Sanchez Fructuoso, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+target+of+rapamycin+signal+inhibitors+could+play+a+role+in+the+treatment+of+BK+polyomavirus+nephritis+in+renal+allograft+recipients.">Mammalian target of rapamycin signal inhibitors could play a role in the treatment of BK polyomavirus nephritis in renal allograft recipients.</a> Transplant Infectious Disease. 13 (6), 584-591 (2011).</li><li>Moens, U., Johansen, T., Johnsen, J. I., Seternes, O. M., Traavik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncoding+control+region+of+naturally+occurring+BK+virus+variants:+sequence+comparison+and+functional+analysis.">Noncoding control region of naturally occurring BK virus variants: sequence comparison and functional analysis.</a> Virus Genes. 10 (3), 261-275 (1995).</li><li>Gosert, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyomavirus+BK+with+rearranged+noncoding+control+region+emerge+in+vivo+in+renal+transplant+patients+and+increase+viral+replication+and+cytopathology.">Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology.</a> Journal of Experimental Medicine. 205 (4), 841-852 (2008).</li><li>Bethge, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sp1+sites+in+the+noncoding+control+region+of+BK+polyomavirus+are+key+regulators+of+bidirectional+viral+early+and+late+gene+expression.">Sp1 sites in the noncoding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression.</a> Journal of Virology. 89 (6), 3396-3411 (2015).</li><li>Gosert, R., Kardas, P., Major, E. O., Hirsch, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearranged+JC+virus+noncoding+control+regions+found+in+progressive+multifocal+leukoencephalopathy+patient+samples+increase+virus+early+gene+expression+and+replication+rate.">Rearranged JC virus noncoding control regions found in progressive multifocal leukoencephalopathy patient samples increase virus early gene expression and replication rate.</a> Journal of Virology. 84 (20), 10448-10456 (2010).</li><li>Bernhoff, E., Gutteberg, T. J., Sandvik, K., Hirsch, H. H., Rinaldo, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cidofovir+inhibits+polyomavirus+BK+replication+in+human+renal+tubular+cells+downstream+of+viral+early+gene+expression.">Cidofovir inhibits polyomavirus BK replication in human renal tubular cells downstream of viral early gene expression.</a> American Journal of Transplantation. 8 (7), 1413-1422 (2008).</li><li>Widera, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+persistent+viremia+is+frequently+followed+by+episodes+of+low-level+viremia.">HIV-1 persistent viremia is frequently followed by episodes of low-level viremia.</a> Medical Microbiology Immunology. , (2017).</li><li>Zhang, S., Cahalan, M. 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N., Crowley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analysis+of+the+complete+genome+of+11+BKV+isolates+obtained+from+allogenic+stem+cell+transplant+recipients+in+Ireland.">Phylogenetic analysis of the complete genome of 11 BKV isolates obtained from allogenic stem cell transplant recipients in Ireland.</a> Journal of Medical Virology. 84 (7), 1037-1048 (2012).</li><li>Dorries, K., Vogel, E., Gunther, S., Czub, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+human+polyomaviruses+JC+and+BK+in+peripheral+blood+leukocytes+from+immunocompetent+individuals.">Infection of human polyomaviruses JC and BK in peripheral blood leukocytes from immunocompetent individuals.</a> Virology. 198 (1), 59-70 (1994).</li><li>Teutsch, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+identification+of+renal+transplant+recipients+with+high+risk+of+polyomavirus-associated+nephropathy.">Early identification of renal transplant recipients with high risk of polyomavirus-associated nephropathy.</a> Medical Microbiology Immunology. 204 (6), 657-664 (2015).</li><li>Basu, S., Campbell, H. 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(41), (2010).</li><li>Korth, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+detection+of+BKPyV+genotypes+II+and+IV+after+renal+transplantation+as+a+simple+tool+for+risk+assessment+for+PyVAN+and+transplant+outcome+already+at+early+stages+of+BKPyV+reactivation.">The detection of BKPyV genotypes II and IV after renal transplantation as a simple tool for risk assessment for PyVAN and transplant outcome already at early stages of BKPyV reactivation.</a> Journal of Clinical Virology. 113, 14-19 (2019).</li><li>Caputo, A., Barbanti-Brodano, G., Wang, E., Ricciardi, R. 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In this article, a commonly used method is presented that allows for the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity. The NCCR activity can be measured simultaneously and does not need lysis of the transfected cells. Furthermore, a relatively large number of cells can be analyzed and the co-transfection of additional markers for normalization of the fluorescence values is not necessary.
A critical part of this method is that the cloned NCCR sh...

Polyomaviruses represent an independent family of small double-stranded DNA (dsDNA) viruses with the Simian virus 40 (SV40) as a prototype species. The primary infection mainly occurs during the childhood, which usually proceed without disease symptoms and usually cause latent infections in immune competent hosts. The BK-polyomavirus (BKPyV) mainly persists in renal tubules cells without causing nephropathologies, however, in case of impairment of the immune-competence after renal transplantation the virus can reactivate and cause severe damages and impaired graft function when reaching a high viremia (1 x 104 BKPyV DNA copies/mL)1,2. In approximately 10% of kidney transplant recipients, reactivation of the BK-polyomavirus (BKPyV) results in a polyomavirus associated nephropathy (PyVAN), which was up to 80% associated with a high risk of renal allograft failures3,4. Since no approved antiviral agents are available, current therapy is based on the reduction of immunosuppression. Interestingly, mTOR inhibitors seem to have an antiviral effect; thus, switching the immunosuppressive therapy to mTOR-based immunosuppression might represent an alternative approach to prevent progression of the BKPyV viremia5,6,7. However, the mTOR-based antiviral mechanism is currently still incompletely understood. Thus, methods measuring the impact of potential antiviral agents in clinically relevant concentrations are required.
The circular genome of the BKPyV consists of approximately 5 kb harboring a non-coding control region (NCCR) that serves as an origin of replication and concomitantly a bidirectional promoter driving the expression of early and late phase mRNA transcripts. Since spontaneously occurring NCCR-rearrangements, deletions, and duplications are found in pathogenic BKPyV8 and significantly accumulated in patients suffering from PVAN5,9, a comparison of archetypical (wt) and re-arranged (rr) NCCR-activities are helpful to characterize viral replicative fitness.
As summarized in Figure 1, this protocol describes a commonly used method to measure BKPyV NCCR transcriptional activity by quantifying the fluorescence of two fluorophores tdTomato and eGFP expressed from a reporter plasmid5,9,10,11. The procedure is performed in the presence of the SV40 large T antigen (lTAg), which allows to analyze the impact of potential antiviral agents on the early and late NCCR-activity separately5. This assay further analyzes the impact of rearrangements on the NCCR activity and comparison with wt-NCCRs5,9. The reporter plasmid harbors the SV40 late polyadenylation signal downstream of each fluorophore open reading frame to ensure comparable and efficient processing of both transcripts for tdTomato and eGFP, respectively. Compared to qRT-PCR based methods5,12, this FACS-based approach represents a low cost and high throughput compatible alternative since no complicated extraction protocols for infected cell culture and no expensive antibodies for immune fluorescence staining are needed. Furthermore, since a defined amount of fluorescent cells are analyzed via flow cytometry, the analysis of cell cycle inhibiting agents is also possible in a quantitative manner.

<table border="0" cellpadding="0" cellspacing="0" style="width:959px;" width="957">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">100 bp ladder</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">N3231</td>
			<td style="width:379px;">Any ladder with a range up to 1 kb can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">2-log ladder</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">N0550</td>
			<td>Any ladder with a range up to 10 kb can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Agar-Agar, Kobe I</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">5210.3</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">AgeI-HF</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">R3552</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Ampicillin Natriumsalz Cellpure</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">HP62.1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Aqua ad iniectabilia</td>
			<td style="width:127px;">Bbraun</td>
			<td style="width:151px;">2351744</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">BD FACSCanto&#8482; II</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">BD FACSDiva</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DAPI</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:151px;">10236276001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DFC450C camera module</td>
			<td style="width:127px;">Leica</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any camera can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DMEM</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">41966-029</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DMIL LED microscope</td>
			<td style="width:127px;">Leica</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any fluorescence microscope can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">DNA Blood Mini Kit</td>
			<td style="width:127px;">Qiagen</td>
			<td style="width:151px;">51104</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">E.coli DH5alpha Competent Cells</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:151px;">18258012</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">FBS Superior</td>
			<td style="width:127px;">MerckMillipore</td>
			<td style="width:151px;">50615</td>
			<td style="width:379px;">Any FBS can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">FlowJo v10.5.3</td>
			<td style="width:127px;">FlowJo, LLC</td>
			<td style="width:151px;"></td>
			<td style="width:379px;">Any flow cytometry software FBS can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Gel Loading Dye, Purple (6X)&#160;</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">B7024</td>
			<td>Any 6x loading dye can be substituted</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HEK293T cells</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:151px;">11268</td>
			<td>These cells constitutively express the simian virus 40 (SV40) large T antigen, and clone 17 was selected specifically for its high transfectability.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HERAcell&#174; 240i CO2 Incubator</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:151px;"></td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">HotStar PCR kit</td>
			<td style="width:127px;">Qiagen</td>
			<td style="width:151px;">203203</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Intas Gel documentation system</td>
			<td style="width:127px;">Intas</td>
			<td style="width:151px;"></td>
			<td>Any visualisation system for stained DNA containing agarose gels can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Low Melt Agarose</td>
			<td style="width:127px;">Biozym</td>
			<td style="width:151px;">850081</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Opti-MEM</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:151px;">31985070</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">pBKV (34-2)</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:151px;">45025</td>
			<td>Plasmid harboring the full-length genome of BKPyV strain Dunlop; was used as a positive control; DNA Seq. Acc.: KP412983</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PBS</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR Cycler MJ Mini 48-Well Personal Cycler</td>
			<td style="width:127px;">Bio-Rad</td>
			<td style="width:151px;">discontinued product</td>
			<td style="width:379px;">Any thermocycler can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR Nucleotide Mix, 10 mM</td>
			<td style="width:127px;">Promega</td>
			<td style="width:151px;">#C1145</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PCR1 and PCR2 Primers</td>
			<td style="width:127px;">metabion</td>
			<td style="width:151px;">not applicable</td>
			<td>Desalted. Dilute to (10 &#181;M) with PCR grade water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">PenStrep (100x)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">15140-122</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Roti&#174;-GelStain</td>
			<td style="width:127px;">Carl Roth</td>
			<td style="width:151px;">3865</td>
			<td>A fluorescence based stain for measuring dsDNA concentration</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">SpeI-HF</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">R3133</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">T4-DNA Ligase</td>
			<td style="width:127px;">NEB</td>
			<td style="width:151px;">M0202</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">TransIT LT1</td>
			<td style="width:127px;">Mirus</td>
			<td style="width:151px;">MIR2300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">Trypsin 0.05% - EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:151px;">25300-054</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:303px;">ZymoPURE II Plasmid Kit</td>
			<td style="width:127px;">Zymo</td>
			<td style="width:151px;">D4201</td>
			<td>can be substituted by any manufacturer</td>
		</tr>
	</tbody>
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measurement of bk-polyomavirus non-coding control region driven transcriptional activity via flow cytometry

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals are currently not available, methods measuring the impact of potential antiviral agents are required. Here, a dual fluorescence reporter that allows the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity was constructed to quantify the impact of potential antiviral drugs on viral gene expression via tdTomato and eGFP expression. In addition, by cloning BKPyV-NCCR amplicons which in this protocol have been exemplarily obtained from the blood-derived DNA of immunocompromised renal transplanted patients, the impact of NCCR-rearrangements on viral gene expression can be determined. Following cloning of the patient derived amplicons, HEK293T cells were transfected with the reporter-plasmids, and treated with potential antiviral agents. Subsequently, cells were subjected to FACS-analysis for measuring mean fluorescence intensities 72 h post transfection. To also test the analysis of drugs that have a potential cell cycle inhibiting effect, only transfected and thus fluorescent cells are used. Since this assay is performed in large T Antigen expressing cells, the impact of early and late expression can be analyzed in a mutually independent manner.

In this representative experiment, the BK-polyomavirus Non-Coding Control Region driven transcriptional activity was measured via flow cytometry. In addition, a mTOR inhibitor, which might be used to treat patients after BKPyV reactivation, was tested for its inhibition of the viral early gene expression. To this end, a dual fluorescence-reporter assay was used as published previously5. The overall workflow scheme of the experimental setup is illustrated in <strong...

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Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals ...

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.

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