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• W1743283402 abstract "The human polyomavirus BK (BKV) causes nephropathy and hemorrhagic cystitis in kidney and bone marrow transplant patients, respectively. The anti-viral cidofovir (CDV) has been used in small case series but the effects on BKV replication are unclear, since polyomaviruses do not encode viral DNA polymerases. We investigated the effects of CDV on BKV(Dunlop) replication in primary human renal proximal tubule epithelial cells (RPTECs). CDV inhibited the generation of viral progeny in a dose-dependent manner yielding a 90% reduction at 40 μg/mL. Early steps such as receptor binding and entry seemed unaffected. Initial large T-antigen transcription and expression were also unaffected, but subsequent intra-cellular BKV DNA replication was reduced by >90%. Late viral mRNA and corresponding protein levels were also 90% reduced. In uninfected RPTECs, CDV 40 μg/mL reduced cellular DNA replication and metabolic activity by 7% and 11% in BrdU and WST-1 assays, respectively. BKV infection increased DNA replication to 142% and metabolic activity to 116%, respectively, which were reduced by CDV 40 μg/mL to levels of uninfected untreated RPTECs. Our results show that CDV inhibits BKV DNA replication downstream of large T-antigen expression and involves significant host cell toxicity. This should be considered in current treatment and drug development. The human polyomavirus BK (BKV) causes nephropathy and hemorrhagic cystitis in kidney and bone marrow transplant patients, respectively. The anti-viral cidofovir (CDV) has been used in small case series but the effects on BKV replication are unclear, since polyomaviruses do not encode viral DNA polymerases. We investigated the effects of CDV on BKV(Dunlop) replication in primary human renal proximal tubule epithelial cells (RPTECs). CDV inhibited the generation of viral progeny in a dose-dependent manner yielding a 90% reduction at 40 μg/mL. Early steps such as receptor binding and entry seemed unaffected. Initial large T-antigen transcription and expression were also unaffected, but subsequent intra-cellular BKV DNA replication was reduced by >90%. Late viral mRNA and corresponding protein levels were also 90% reduced. In uninfected RPTECs, CDV 40 μg/mL reduced cellular DNA replication and metabolic activity by 7% and 11% in BrdU and WST-1 assays, respectively. BKV infection increased DNA replication to 142% and metabolic activity to 116%, respectively, which were reduced by CDV 40 μg/mL to levels of uninfected untreated RPTECs. Our results show that CDV inhibits BKV DNA replication downstream of large T-antigen expression and involves significant host cell toxicity. This should be considered in current treatment and drug development. Polyomavirus BK (BKV) infects up to 90% of the general population and establishes latent infection in the renourinary tract (1Hirsch HH Steiger J Polyomavirus BK.Lancet Infect Dis. 2003; 3: 611-623Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Spontaneous reactivation and shedding of BKV have been observed in 5-10% of immunocompetent individuals. In immunosuppressed individuals, the rate of urinary shedding increases to 20-60% with viral loads > 107copies/mL (1Hirsch HH Steiger J Polyomavirus BK.Lancet Infect Dis. 2003; 3: 611-623Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar,2Polo C Perez JL Mielnichuck A Fedele CG Niubo J Tenorio A Prevalence and patterns of polyomavirus urinary excretion in immunocompetent adults and children.Clin Microbiol Infect. 2004; 10: 640-644Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Two major diseases have been associated with BKV, namely polyomavirus-associated nephropathy after kidney transplantation and hemorrhagic cystitis following bone marrow transplantation (1Hirsch HH Steiger J Polyomavirus BK.Lancet Infect Dis. 2003; 3: 611-623Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Polyomavirus-associated nephropathy results from high-level replication of BKV in renal tubular epithelial cells and leads to reduced kidney function and graft loss (3Randhawa PS Finkelstein S Scantlebury V et al.Human polyoma virus-associated interstitial nephritis in the allograft kidney.Transplantation. 1999; 67: 103-109Crossref PubMed Scopus (480) Google Scholar, 4Hirsch HH Knowles W Dickenmann M et al.Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients.N Engl J Med. 2002; 347: 488-496Crossref PubMed Scopus (992) Google Scholar, 5Ramos E Drachenberg CB Portocarrero M et al.BK virus nephropathy diagnosis and treatment: Experience at the University of Maryland Renal Transplant Program.Clin Transpl. 2002; : 143-153PubMed Google Scholar, 6Fishman JA BK virus nephropathy-polyomavirus adding insult to injury.N Engl J Med. 2002; 347: 527-530Crossref PubMed Scopus (130) Google Scholar). The primary treatment is reduction of immunosuppression although this might increase the risk of graft rejection (7Hirsch HH Brennan DC Drachenberg CB et al.Polyomavirus-associated nephropathy in renal transplantation: Interdisciplinary analyses and recommendations.Transplantation. 2005; 79: 1277-1286Crossref PubMed Scopus (816) Google Scholar). Hemorrhagic cystitis is associated with high-level replication of BKV in urothelial cells, and current treatment is symptomatic with pain relief and bladder irrigation. There are no drugs with proven efficacy for BKV replication (8Rinaldo CH Hirsch HH Antivirals for the treatment of polyomavirus BK replication.Expert Rev Anti Infect Ther. 2007; 5: 105-115Crossref PubMed Scopus (102) Google Scholar). Cidofovir (CDV), a nucleoside analogue of deoxy cytidine mono phosphate (dCMP), has been sporadically used for the treatment of BKV replication in patients with nephropathy or hemorrhagic cystitis (8Rinaldo CH Hirsch HH Antivirals for the treatment of polyomavirus BK replication.Expert Rev Anti Infect Ther. 2007; 5: 105-115Crossref PubMed Scopus (102) Google Scholar, 9Benavides CA Pollard VB Mauiyyedi S Podder H Knight R Kahan BD BK virus-associated nephropathy in sirolimus-treated renal transplant patients: Incidence, course, and clinical outcomes.Transplantation. 2007; 84: 83-88Crossref PubMed Scopus (70) Google Scholar, 10Rajpoot DK Gomez A Tsang W Shanberg A Ureteric and urethral stenosis: A complication of BK virus infection in a pediatric renal transplant patient.Pediatr Transplant. 2007; 11: 433-435Crossref PubMed Scopus (36) Google Scholar, 11Cheerva AC Raj A Bertolone SJ Bertolone K Silverman CL BK virus-associated hemorrhagic cystitis in pediatric cancer patients receiving high-dose cyclophosphamide.J Pediatr Hematol Oncol. 2007; 29: 617-621Crossref PubMed Scopus (17) Google Scholar, 12Andrei G Fiten P Goubau P et al.Dual infection with polyomavirus BK and acyclovir-resistant herpes simplex virus successfully treated with cidofovir in a bone marrow transplant recipient.Transpl Infect Dis. 2007; 9: 126-131Crossref PubMed Scopus (24) Google Scholar) However, it is not clear if the usually favorable outcome of these reports can be attributed to the anti-polyomavirus activity of the drug or rather involved recovery of BKV-specific immunity. CDV is licensed to treat ganciclovir resistant cytomegalovirus (CMV) retinitis in patients with HIV/AIDS (13De Clercq E Acyclic nucleoside phosphonates: Past, present and future. Bridging chemistry to HIV, HBV, HCV, HPV, adeno-, herpes-, and poxvirus infections: The phosphonate bridge.Biochem Pharmacol. 2007; 73: 911-922Crossref PubMed Scopus (141) Google Scholar), but an important drawback of systemic CDV use is its nephrotoxicity (1Hirsch HH Steiger J Polyomavirus BK.Lancet Infect Dis. 2003; 3: 611-623Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 14Ho ES Lin DC Mendel DB Cihlar T Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1.J Am Soc Nephrol. 2000; 11: 383-393Crossref PubMed Google Scholar, 15Izzedine H Launay-Vacher V Deray G Renal tubular transporters and antiviral drugs: An update.AIDS. 2005; 19: 455-462Crossref PubMed Scopus (55) Google Scholar, 16Ortiz A Justo P Sanz A et al.Tubular cell apoptosis and cidofovir-induced acute renal failure.Antivir Ther. 2005; 10: 185-190Crossref PubMed Google Scholar, 17Cihlar T Ho ES Lin DC Mulato AS Human renal organic anion transporter 1 (hOAT1) and its role in the nephrotoxicity of antiviral nucleotide analogs.Nucleosides Nucleotides Nucleic Acids. 2001; 20: 641-648Crossref PubMed Scopus (220) Google Scholar, 18Miller DS Nucleoside phosphonate interactions with multiple organic anion transporters in renal proximal tubule.J Pharmacol Exp Ther. 2001; 299: 567-574PubMed Google Scholar, 19Wolf DL Rodriguez CA Mucci M Ingrosso A Duncan BA Nickens DJ Pharmacokinetics and renal effects of cidofovir with a reduced dose of probenecid in HIV-infected patients with cytomegalovirus retinitis.J Clin Pharmacol. 2003; 43: 43-51Crossref PubMed Scopus (36) Google Scholar). So far, only two in vitro studies have examined the effect of CDV on BKV replication. The first study of human embryonic lung fibroblasts (WI-38 cells) found that CDV concentrations of 36.3 ± 11.7 μg/mL reduced intra-cellular BKV DNA levels by 50% after 7 days, while higher CDV concentrations of 63.9 ± 17.2 μg/mL also reduced cellular DNA by 50% (20Farasati NA Shapiro R Vats A Randhawa P Effect of leflunomide and cidofovir on replication of BK virus in an in vitro culture system.Transplantation. 2005; 79: 116-118Crossref PubMed Scopus (137) Google Scholar). In the second study, seven BKV isolates from urine were inoculated onto human embryonic lung fibroblasts (HEL) and scoring the microscopic appearance of cytopathic effects until 30 days. At lower BKV inocula, termed 10 × TCID50, CDV concentrations of 31.25 μg/mL inhibited the cytopathic effect of five isolates, whereas two isolates required higher CDV concentrations (21Leung AY Chan MT Yuen KY et al.Ciprofloxacin decreased polyoma BK virus load in patients who underwent allogeneic hematopoietic stem cell transplantation.Clin Infect Dis. 2005; 40: 528-537Crossref PubMed Scopus (182) Google Scholar). At higher BKV inocula, only two strains seemed inhibited by CDV. In herpes and poxvirus the CDV inhibition results from the diphosphorylated CDV, which has higher affinity for the viral DNA polymerase than for the cellular enzyme (22Magee WC Aldern KA Hostetler KY Evans DH Cidofovir and (S)-HPMPA are highly effective inhibitors of vaccinia virus DNA polymerase when incorporated into the template strand.Antimicrob Agents Chemother. 2008; 52: 586-597Crossref PubMed Scopus (73) Google Scholar, 23Ho HT Woods KL Bronson JJ De BH Martin JC Hitchcock MJ Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine.Mol Pharmacol. 1992; 41: 197-202PubMed Google Scholar, 24Neyts J Snoeck R Schols D Balzarini J De Clercq E Selective inhibition of human cytomegalovirus DNA synthesis by (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [(S)-HPMPC] and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG).Virology. 1990; 179: 41-50Crossref PubMed Scopus (86) Google Scholar) and blocks excision repair of the growing viral genome. The effects of CDV on BKV are less clear since polyomaviruses do not encode a viral DNA polymerase. Here we report on a detailed study of the effects on CDV on the BKV lifecycle in primary human renal proximal tubule epithelial cells (RPTECs) as well as the effects on host cell DNA replication and metabolism. Primary human renal proximal tubule epithelial cells (RPTECs) (Lonza, http://www.lonzabioscience.com) were propagated as described by the manufacturer. All experiments were performed with RPTECs passage 4. BKV(Dunlop) supernatants from infected Vero cells were used with a multiplicity of infection (MOI) determined on HUV-EC-C (ATCC CRL1730) (25Grinde B Gayorfar M Rinaldo CH Impact of a polyomavirus (BKV) infection on mRNA expression in human endothelial cells.Virus Res. 2007; 123: 86-94Crossref PubMed Scopus (35) Google Scholar). About ∼60% confluent RPTECs were infected with BKV(Dunlop) at MOI of ∼1. After 2-h incubation at 37°C the virus was replaced with fresh medium with or without CDV (VISTIDE 75 mg/mL, anhydrous CDV, Pfizer Enterprises SARL, Luxembourg). CDV was freshly diluted in medium to 5, 10, 20, 40 and 80 μg/mL and added to BKV-infected and uninfected RPTECs. DNA synthesis was quantified by the colorimetric measurement of BrdU incorporation into DNA in proliferating cells using the Cell proliferation ELISA, BrdU kit (Roche, http://www.roche-applied-science.com). The metabolic activity was monitored by the colorimetric WST-1 assay (Roche) of the mitochondrial dehydrogenases in viable cells. For both assays, RPTECs were seeded in 96 well plates and infected with BKV (Dunlop) or mock infected as described above. BrdU incorporation and WST-1 cleavage was measured at 24, 48, 72 and 96 h p.i. according to the manufacturer’s protocol. Absorbance at 370 nm (sample) and at 450 nm (background) was determined 15 min after addition of the substrate (Spectramax plus, Molecular Devices). The WST-1 cleavage product was measured at 450 nm (sample) and at 650 nm (background). WST-1 plus medium alone served as blank. Total cellular RNA was harvested 24, 48, 72 and 96 h p.i. using the mirVana PARIS kit (Ambion, Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. RNA samples were treated with DNase turbo (Ambion) to remove residual DNA before RNA concentration and assayed by Nanodrop (Nanodrop Technologies, Thermo Fisher Scientific, Waltham, MA), and by microcapillary electrophoresis on a Bioanalyser 2100 (Agilent Technologies, Santa Clara, CA). cDNA was generated from 280 ng RNA per sample using the High Capacity cDNA Kit (Applied Biosystems). To assay extracellular DNA, cell culture supernatants were harvested at 24, 48, 72 and 96 h p.i. and frozen in −80°C until automatic extraction by a robot (GenoM-48, Qiagen, http://www.qiagen.com). Intra-cellular DNA was extracted from cells using the Qiagen DNA mini kit. Cells were first washed, trypsinized and pelleted at 220 g for 10 min, then resuspended in phosphate-buffered saline (PBS) and frozen at −80°C until extraction. Cells were lysed in Cell Disruption buffer (mirVana PARIS kit, Ambion) and stored at −80°C until separation on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto Immobilon-FL PVDF membrane (Millipore, http://www.millipore.com), blocked with blocking buffer (Odyssey, http://www.licor.com) and incubated with polyclonal rabbit sera anti-large T-antigen (1:2000) (26Hey AW Johnsen JI Johansen B Traavik T A two fusion partner system for raising antibodies against small immunogens expressed in bacteria.J Immunol Methods. 1994; 173: 149-156Crossref PubMed Scopus (30) Google Scholar), anti-VP1 (1: 10000) (25Grinde B Gayorfar M Rinaldo CH Impact of a polyomavirus (BKV) infection on mRNA expression in human endothelial cells.Virus Res. 2007; 123: 86-94Crossref PubMed Scopus (35) Google Scholar) and anti-agnoprotein (1:10000) (26Hey AW Johnsen JI Johansen B Traavik T A two fusion partner system for raising antibodies against small immunogens expressed in bacteria.J Immunol Methods. 1994; 173: 149-156Crossref PubMed Scopus (30) Google Scholar) followed by anti-rabbit infrared dye-labeled secondary antibodies (IR Dye 800, Rockland, http://www.rockland-inc.com) (1:5000) before detection with Licor Odyssey Infrared detection system. Membranes were stripped in NewBlot Stripping Buffer PVDF (Odyssey) before reprobing with monoclonal anti-GAPDH (1:5000) (ab8245, Abcam, http://www.abcam.com) and anti-mouse (Alexa Fluor 680, Invitrogen, http://www.invitrogen.com). Three days post-infection, cells were washed in PBS, fixed in methanol and blocked with 3% serum in goat-serum in PBS for 30 min and then treated as earlier described (27Leuenberger D Andresen PA Gosert R et al.Human Polyomavirus type 1 (BK virus) Agnoprotein is abundantly expressed, but immunologically ignored.Clin Vaccine Immunol. 2007; 14: 959-968Crossref PubMed Scopus (42) Google Scholar). In addition, we used polyclonal rabbit serum anti-VP1 (1:800). To quantify intra-cellular or extracellular BKV DNA load, a quantitative PCR (qPCR) with primers and probe targeting the large T-antigen gene was used (Table 1). Reactions were performed in a total volume of 25 μL using 2× Taqman Fast Universal PCRmaster mix (Applied Biosystems), 5 μL DNA template, 300 nm of each primer and 125 nm probe. For normalization of intra-cellular BKV DNA each sample was analyzed in parallel by the qPCR for the gene for aspartoacylase (ACY) (28Randhawa PS Vats A Zygmunt D et al.Quantitation of viral DNA in renal allograft tissue from patients with BK virus nephropathy.Transplantation. 2002; 74: 485-488Crossref PubMed Scopus (95) Google Scholar) to correct for cellular DNA (Table 1). For ACY qPCR, 300 nm of each primer, and 250 nm probe were used. Amplification was performed in 96 well PCR plates using the 7500 HT cycler (Applied Biosystems) with the following PCR program: denaturation at 95°C, 20 and 45 cycles of 95°C, 3; 60°C, 30.Table 1Primers and probes usedGene/Transcriptrimer and probe sequences (5’ to 3’)DyeFragment lengthReferenceT-antigenF: TAG GTG CCA ACC TAT GGA ACA GAR: RMR GGA AAG TCY TTR GGG TCT TCT ACCFAM185 bpP: TGT TGA GTG TTG AGA ATC TGC TGT TGC TTC TTCAsparto-acylase (ACY)F: CCC TGC TAC GTT TAT CTG ATT GAGR: CCC ACA GGA TAC TTG GCT ATG GTAMRA-77 bp(28)P: CCT TCC CTC AAA TAT GCG ACC ACT CGT-antigen cDNAF: ACT CCC ACT CTT CTG TTC CAT AGGR: TCA TCA GCC TGA TTT TGG AAC CTFAM68 bpP: TTG GCA CCT CTG AGC TACVP1/Agno cDNAF: GGC CTC TTT GTA AAG CTG ATA GCR: CTG TTG TGT TCC AGA GCT GTT AGT AFAM83 bpP: CAG CTG CTG ATA TTT GhuHPRT cDNAP: TGG TCA AGG TCG CAA GCT TGC TGG TFAMhttp://www.appliedbiosystems.comF = forward primer; R = reverse primer; P = probe. Open table in a new tab F = forward primer; R = reverse primer; P = probe. To quantify BKV early gene (T-antigen) and late (VP1 and agnoprotein) gene transcription expression, reverse transcription-quantitative PCR (RT-qPCR) was performed (Table 1). RT-qPCR was performed in 25 μL using 2× Taqman Fast Universal PCR master mix, 5 μL cDNA template and 20× Expression Assay Mix (Applied Biosystems). Results are presented as the changes (n-fold) in T-antigen- or late-transcript levels, compared to untreated sample 24 h p.i. The 2−∆∆C(T) method (29Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (127074) Google Scholar) were used for normalization to the levels of endogenous control transcript human Hypoxanthine PhosphoRibosyl-Transerase (huHPRT). To identify host cell control transcripts, we used the TaqMan Endogenous Control Plate (Applied Biosystems) with 11 human housekeeping genes. cDNA were made from: (a) BKV-infected CDV (80 μg/mL) treated RPTECs 24 h p.i., (b) BKV-infected CDV (80 μg/mL) treated RPTECs 48 h p.i., (c) BKV-infected untreated RPTECs 48 h p.i. and (d) Uninfected untreated RPTECs 48 h p.i. used as calibrator sample. Amplification was performed in duplicate according to the manufacturer’s protocol. The results were presented as ∆Ct and 5 genes had ∆Ct < than 0.5 with huHPTR showing the least variation. To examine the replication cycle of BKV(Dunlop) strain in RPTECs, cells were infected at MOI ∼1.0. Supernatants and cells were harvested 2-96 h p.i., and the corresponding extracellular and intra-cellular BKV load was measured. Increases of intra-cellular BKV load were detected from 24 h p.i onward while extracellular BKV loads increased from 48 h p.i. (Figure 1A). Thus, completion of the first lifecycle of BKV(Dunlop) in RPTECs takes approximately 48-72 h. To investigate the effect of CDV on BKV progeny, increasing concentrations of CDV were added 2 h p.i. and supernatants harvested at 72 h p.i. We observed that CDV reduced the extracellular BKV load in a concentration-dependent manner (Figure 1B). CDV 5 μg/mL reduced the BKV load by only 66% or 0.47 log. CDV 40 μg/mL reduced the BKV load by an average of 90% therefore defining the inhibitory concentration IC90. Immunofluorescence staining at 72 h p.i. demonstrated less agnoprotein expressing cells at 40 and 80 μg/mL, whereas 5 μg/mL had no discernable effect compared to untreated cells (Figure 1C). To investigate whether or not the inhibitory effect of CDV was dependent on levels of input virus, RPTECs were infected with decreasing MOI of BKV and BKV loads in supernatants 72 h p.i. were examined. Diluting the input MOI over three orders of magnitude, a reduction of 94-98% was observed (Figure 1D). Inspection of RPTECs by phase contrast microscopy did not reveal any signs of impaired viability during the 4-day exposure to CDV 40 μg/mL. To use more sensitive assays we investigated host cell DNA replication and metabolic activity using BrdU incorporation and WST-1 assays in BKV-infected and uninfected RPTECs. Compared to uninfected untreated RPTECs, BKV infection by itself increased cellular DNA replication and metabolic activity to 142 and 116%, respectively (Figure 2A). Addition of CDV reduced both DNA replication and metabolic activity of BKV-infected RPTECs in a concentration-dependent manner (Figure 2A). Compared to BKV-infected untreated RPTECs, CDV at 5 to 80 μg/mL decreased DNA replication by 13% to 33%, respectively. CDV concentrations up to 20 μg/mL did not affect the metabolic activity, but a 10-40% decrease was seen with 20-80 μg/mL. Of note, CDV 40 μg/mL reduced DNA replication in BKV-infected cells by 36% and metabolic activity by 23%, to activities similar to untreated uninfected cells. In uninfected cells, CDV 40 μg/mL also reduced BrdU incorporation and metabolic activity with 7 and 11%, respectively. When we measured BrdU incorporation and metabolic activity from 24-96 h p.i., DNA replication was decreased at all time points, while effects on metabolic activity became prominent after 48 h p.i. (Figure 2B). Taken together, CDV significantly reduced host cell replicative and metabolic activity. To investigate whether CDV affected early steps of the BKV lifecycle, CDV 40 μg/mL was added 2 h before, together with and 2 h after adding BKV to RPTECs. No significant difference in the extracellular BKV loads were observed at 72 h p.i. (Figure 3) suggesting that CDV did not significantly interfere with early steps such as receptor binding and viral entry. To study the effects of CDV on BKV early gene expression, we compared T-antigen mRNA levels in CDV-treated and untreated RPTECs at 24-96 h p.i. by RT-qPCR. Large T-antigen mRNA levels were normalized to the housekeeping gene huHPRT and presented as changes (n-fold) of the levels found in untreated cells at 24 h p.i. No differences in T-antigen transcripts were seen in CDV-treated and untreated cells at 24 h p.i. At 48 and 72 h p.i., a moderate reduction of 2.3- and 1.5-fold of T-antigen transcripts was noted, respectively (Figure 4A). When protein levels of large T-antigen were compared, we found no differences of T-antigen levels in CDV-treated and untreated cells 24 h p.i. At 48 and 72 h p.i., however, a five- and three-fold reduction was seen (Figure 4B). These results indicated that initial large T-antigen expression up to 24 h p.i. was not affected by CDV. From 48 h to 72 h p.i., however, a moderate reduction in viral early gene expression became discernible. We concluded that CDV must decrease BKV progeny in supernatants at a step downstream of the initial BKV early gene expression. To investigate whether the next step of the BKV lifecycle BKV genome replication was affected by CDV, we measured intra-cellular BKV load at 24-96 h p.i. by qPCR. The intra-cellular BKV load was normalized to the cell number using the aspartoacylase (ACY) gene as described (30Randhawa P Shapiro R Vats A Quantitation of DNA of polyomaviruses BK and JC in human kidneys.J Infect Dis. 2005; 192: 504-509Crossref PubMed Scopus (53) Google Scholar). Compared to untreated RPTECs, CDV 40 μg/mL reduced the intra-cellular BKV load by more than 99% at 48 h, 72 h and 96 h p.i., whereas only a small effect was seen at 24 h p.i. (Figure 5). Thus, intra-cellular BKV genome replication is the first step of the BKV lifecycle where we could identify a significant inhibitory effect of CDV. This step is known to require large T-antigen expression, which also increases viral late gene expression by increasing the DNA templates for late gene transcription and by activating transcription from the late promoter (31Cole CN Polyomavirinae: The viruses and their replication.in: Fields BN Knipe DM Howley PH Fields Virology. Lippincott-Raven, New york1996: 1997-2043Google Scholar). To study the effect of CDV on BKV late gene transcription, VP1 and agnoprotein mRNA expression was measured by RT-qPCR 48-96 h p.i. and normalized to huHPRT transcripts as described above. The primers and probe for late mRNA expression are located in the VP1 gene but detect mRNA for VP1 and agnoprotein because the transcripts are bicictronic. In untreated cells, late transcripts increased from 24 h p.i. more than 1000-, 5000- and 8000-fold at 48 h, 72 h and 96 h p.i., respectively (Figure 6A). With CDV 40 μg/mL treatment the corresponding increase was only 100-, 700- and 800-fold. Thus the expression was 85-90% reduced in CDV-treated cells. When we examined viral late protein expression, a significant reduction of VP1 and agnoprotein was observed in CDV-treated RPTECs. At 72 h p.i., VP1 and agnoprotein were both found to be 90% reduced in CDV-treated cells compared to untreated controls (Figure 6B). We conclude that CDV significantly reduces late protein expression. Given the significant difference between early and late gene expression, we examined large T-antigen, agnoprotein and VP1 expression at the single-cell level by immunofluorescence staining at 72 h p.i. We found a slight reduction in large T-antigen positive cells of 8% compared to a large reduction of 90% in agnoprotein-positive cells (Figure 6C). Similar results were found for VP1 staining (results not shown). Thus, the overall inhibition of CDV on BKV late gene expression was detectable at the single-cell level. Interestingly, immunofluorescence revealed some cells in the CDV treated culture expressing agnoprotein at levels comparable to untreated cells. Even when increasing the CDV concentration up to 300 μg/mL, some cells seemed refractory to exposure of CDV. To examine the effect of CDV on BKV progeny over time, supernatants of treated and untreated cells were harvested at the indicated time points. The reduction of BKV loads in supernatants reached 95% (1.74 × 109 Geq/mL) at 96 h p.i. (Figure 7A). We next examined whether or not CDV had a persistent or transient effect on BKV replication in RPTECs. Removal of CDV containing medium after 24 h reduced BKV loads in the 96-h supernatants by only 0.89 log Geq/mL. CDV removal after 48 h caused a reduction of 1.62 log Geq/mL (Figure 7B) while 96-h treatment gave a 1.98 log Geq/mL reduction. We concluded that CDV exposure the first 24 h had only a limited inhibitory effect that further increased when the drug was present for 48 h or 96 h. High-level BKV replication is the common hallmark of hemorrhagic cystitis and nephropathy jeopardizing the outcome of hemopoietic stem cell transplantation and kidney transplantation in 1-10% of affected patients. The urgent need of anti-viral drugs, has led to the tentative use of CDV in small uncontrolled case series. However, given the potential for serious, partly irreversible side effects, particularly for kidneys, there is a clear need to better characterize the effects of CDV on BKV replication in clinically relevant experimental systems. In this study, we report that CDV inhibits BKV replication in primary human renal tubular epithelial cells at a step downstream of large T-antigen expression and is associated with significant host cell toxicity. With regard to BKV replication, we found that CDV at 40 μg/mL caused a 90% reduction of extracellular BKV loads. Presence of CDV 2 h before and during incubation of RPTECs with BKV did not affect rate of inhibition suggesting that early steps of the viral replication cycle as receptor binding and entry were not affected. In fact, our detailed study of the BKV replication cycle revealed that the initial early gene expression up to 24 h p.i. was not significantly reduced by CDV as measured by large T-antigen mRNA and protein levels. However, CDV significantly inhibited intra-cellular BKV genome replication. This step occurs after 24 h p.i. and is dependent on prior large T-antigen expression providing DNA helicase functions as well as recruiting host cell replication factors. This notion was also supported by wash-out experiments indicating that the presence of CDV was required for more than 24 h to reach near-maximal inhibition of the first replication cycle. In line with the known role of amplification of DNA templates for late viral gene transcription by viral DNA replication (31Cole CN Polyomavirinae: The viruses and their replication.in: Fields BN Knipe DM Howley PH Fields Virology. Lippincott-Raven, New york1996: 1997-2043Google Scholar), CDV reduced overall late mRNA and proteins levels by about 90%. Importantly, immunofluorescent staining allowed the analysis of viral protein expression at the single-cell level indicating that the nuclear large T-antigen expression was largely similar to untreated cells, whereas the late agnoprotein expression was virtually undetectable in at least 90% of treated cells. Interestingly, about one-tenth of CDV-treated cells seemed refractory to inhibition and this could not be overcome by higher concentrations. The underlying mechanism of this seemingly stochastic resistance is presently unclear and requires further study. Most likely, these refractory cells are at least partly responsible for generating residual BKV progeny, at least in tissue culture. With regard to the host cells, we observed that CDV significantly inhibited the overall cellular DNA replication and metabolic activity. This effect was more pronounced in BKV-infected than in uninfected RPTECs. Interestingly, both, DNA replication and metabolic activity was increased following BKV infection which is a consequence of large T-antigen inactivating the tumor-suppressor proteins Rb and p53 and stimulating cell progression into S-phase (32Harris KF Christensen JB Imperiale MJ BK virus large T antigen: Interactions with the retinoblastoma family of tumor suppressor proteins and effects on cellular growth control.J Virol. 1996; 70: 2378-2386Crossref PubMed Google Scholar). Thus, CDV at 40 μg/mL decreased BrdU and WST-1 activities to levels seen in uninfected untreated cells. These data document significant toxicity of CDV on human RPTECs, which was not visible by microscopic evaluation. Part of the toxic effects by CDV are likely to come from its incorporation into host cell genomic DNA as reported in a study of human papillomaviruses that, akin to BKV, lack a viral DNA polymerase (33Spanos WC El-Deiry M Lee JH Cidofovir incorporation into human keratinocytes with episomal HPV 16 results in nonselective cytotoxicity.Ann Otol Rhinol Laryngol. 2005; 114: 840-846Crossref PubMed Scopus (13) Google Scholar). The IC90 of 40 μg/mL in RPTECs is similar to the IC50 reported for the inhibition of BKV replication in lung fibroblast lines (20Farasati NA Shapiro R Vats A Randhawa P Effect of leflunomide and cidofovir on replication of BK virus in an in vitro culture system.Transplantation. 2005; 79: 116-118Crossref PubMed Scopus (137) Google Scholar,21Leung AY Chan MT Yuen KY et al.Ciprofloxacin decreased polyoma BK virus load in patients who underwent allogeneic hematopoietic stem cell transplantation.Clin Infect Dis. 2005; 40: 528-537Crossref PubMed Scopus (182) Google Scholar). We suspect that this is partly the result of the active transport system for organic compounds present in RPTEC (14Ho ES Lin DC Mendel DB Cihlar T Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1.J Am Soc Nephrol. 2000; 11: 383-393Crossref PubMed Google Scholar, 15Izzedine H Launay-Vacher V Deray G Renal tubular transporters and antiviral drugs: An update.AIDS. 2005; 19: 455-462Crossref PubMed Scopus (55) Google Scholar, 16Ortiz A Justo P Sanz A et al.Tubular cell apoptosis and cidofovir-induced acute renal failure.Antivir Ther. 2005; 10: 185-190Crossref PubMed Google Scholar, 17Cihlar T Ho ES Lin DC Mulato AS Human renal organic anion transporter 1 (hOAT1) and its role in the nephrotoxicity of antiviral nucleotide analogs.Nucleosides Nucleotides Nucleic Acids. 2001; 20: 641-648Crossref PubMed Scopus (220) Google Scholar, 18Miller DS Nucleoside phosphonate interactions with multiple organic anion transporters in renal proximal tubule.J Pharmacol Exp Ther. 2001; 299: 567-574PubMed Google Scholar) yielding higher intra-cellular CDV concentration than in lung fibroblasts. It is not clear if CDV has a similar effect on DNA replication and metabolic activity in lung fibroblasts, but our study emphasizes the importance of choosing pathologically relevant in vitro models. In view of the IC90 of 40 μg/mL, do our findings support clinical use of CDV? In a clinical study by Kuypers and colleagues (34Kuypers DR Vandooren AK Lerut E et al.Adjuvant low-dose cidofovir therapy for BK polyomavirus interstitial nephritis in renal transplant recipients.Am J Transplant. 2005; 5: 1997-2004Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), CDV peak concentrations in serum of eight nephropathy patients treated with reduced immunosuppression and low-dose CDV (0,5 mg/kg or 1,0 mg/kg), were found to be 0,77-3,07 μg/mL. This is 13- to 52-times less than the IC90 determined here in vitro. However, active uptake of CDV into tubular epithelial cells may allow for some increased intra-cellular concentration over what is found in serum. Assuming that uptake is similarly active in RPTECs in vitro, we note that we observed no relevant inhibition of BKV replication or late gene expression at CDV concentration 5 μg/mL. We wonder, therefore, how much of the favorable results in that study can in fact be attributed to CDV. When CDV was administered to HIV patients at 7.5 mg/kg with concomitant high-dose probenicid, maximal CDV concentrations of 43 μg/mL could be reached, but probenicid may prevent uptake into tubular epithelial cells (35Cundy KC Petty BG Flaherty J et al.Clinical pharmacokinetics of cidofovir in human immunodeficiency virus-infected patients.Antimicrob Agents Chemother. 1995; 39: 1247-1252Crossref PubMed Scopus (181) Google Scholar). Can a 90% inhibition be sufficient to clear the virus in view of the high-level BKV replication of 107-1010 copies/mL in patients with nephropathy or with hemorrhagic cystitis? Results from mathematical modeling of BKV replication in urothelial and renal tubular epithelial cells suggest that a more than 80% reduction of renal BKV replication must be maintained for up to 10 weeks to observe clearing of plasma and urine viral loads (36Hirsch HH, Gosert R, Funk GA. The 3rd International Transplant Infectious Disease Conference September 29-30, 2007, Prague, Czech Republic, Available from: http://www.intmedpress.com/tid/Google Scholar). Based on these data, long-term maintenance of inhibitory CDV concentrations may be necessary. Thus, we see at least three caveats. First, long-term exposure and intra-cellular accumulation increases the risk of nephrotoxicity. Even though uninfected RPTECs seem less affected than BKV-infected cells, considerable toxicity must be expected from CDV acting on proliferating tubular epithelial cells replacing the approximately 106-107 cells lost each day due to fast BKV replication dynamics (37Funk GA Steiger J Hirsch HH Rapid dynamics of polyomavirus type BK in renal transplant recipients.J Infect Dis. 2006; 193: 80-87Crossref PubMed Scopus (81) Google Scholar). Second, the immunofluorescence data shown here revealed that CDV inhibition was only partial, with some cells being completely refractory which may allow for continuous replication and emerging resistance. Third, in cells with inhibited BKV replication, the large T-antigen was expressed and these cells may continue the BKV lifecycle once CDV concentrations become insufficient as observed in our wash-out experiments. Transient inhibition by CDV has been reported for papillomavirus (33Spanos WC El-Deiry M Lee JH Cidofovir incorporation into human keratinocytes with episomal HPV 16 results in nonselective cytotoxicity.Ann Otol Rhinol Laryngol. 2005; 114: 840-846Crossref PubMed Scopus (13) Google Scholar), but not for herpes and vacciniavirus, which encode viral DNA polymerases (23Ho HT Woods KL Bronson JJ De BH Martin JC Hitchcock MJ Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine.Mol Pharmacol. 1992; 41: 197-202PubMed Google Scholar,24Neyts J Snoeck R Schols D Balzarini J De Clercq E Selective inhibition of human cytomegalovirus DNA synthesis by (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [(S)-HPMPC] and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG).Virology. 1990; 179: 41-50Crossref PubMed Scopus (86) Google Scholar,38Neyts J De Clercq E Efficacy of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine for the treatment of lethal vaccinia virus infections in severe combined immune deficiency (SCID) mice.J Med Virol. 1993; 41: 242-246Crossref PubMed Scopus (96) Google Scholar). These caveats do not preclude the clinical use of CDV, but should be factored into the assessment of the drug. In conclusion, our study of CDV effects on BKV infection of primary human RPTECs demonstrate inhibition of BKV DNA replication downstream of initial large T-antigen expression with significant host cell toxicity. This needs to be considered in current treatment strategies and the development of future drugs. This research was supported by Fondet for forskning om nyresykdommer og organtransplantasjon, Landsforeningen for nyrepasienter og transplanterte, Norway." @default.
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• W1743283402 title "Cidofovir Inhibits Polyomavirus BK Replication in Human Renal Tubular Cells Downstream of Viral Early Gene Expression" @default.
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