1
1-O-hexadecyloxypropyl cidofovir (CMX001) effectively inhibits polyomavirus BK 1
replication in primary human renal tubular cells 2
3
Running title: CMX001 inhibition of BKV replication in kidney cells 4
5
Christine Hanssen Rinaldo 1, Rainer Gosert 2, Eva Bernhoff 1, Solrun Finstad 1and Hans H. 6
Hirsch 2,3* 7
1Department of Microbiology and Infection Control, University Hospital of North Norway, 8
Tromsø, Norway 9
2Transplantation Virology, Institute for Medical Microbiology, Department of Biomedicine, 10
University of Basel, Basel, Switzerland 11
3Infectious Diseases & Hospital Epidemiology, University Hospital Basel, Basel, Switzerland 12
13
*Corresponding author: 14
Hans H. Hirsch, MD MS 15
Transplantation Virology, Institute for Medical Microbiology, Department of Biomedicine, 16
University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland, 17
Phone +41 61 267 3262 18
Fax +41 61 267 3283 19
20
21
Word counts 22
Abstract: 250 23
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00974-10 AAC Accepts, published online ahead of print on 16 August 2010
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ABSTRACT 24
Antiviral drugs for treating BKV replication in polyomavirus-associated nephropathy or -25
hemorrhagic cystitis are of considerable clinical interest. Unlike cidofovir, the lipid conjugate 26
1-O-hexadecyloxypropyl-cidofovir (CMX001) is orally available and has not caused 27
detectable nephrotoxicity in rodent models or human studies to date. Primary human renal 28
proximal tubular epithelial cells were infected with BKV-Dunlop and CMX001 was added 2h 29
post-infection (hpi). Intracellular and extracellular BKV DNA load was determined by 30
quantitative PCR. Viral gene expression was examined by quantitative reverse transcription 31
PCR, western blotting, and immunofluorescence microscopy. We also examined host cell 32
viability, proliferation, metabolic activity, and DNA replication. Titration of CMX001 identified 33
0.31 µM as the 90% effective concentration (EC90) for reducing the extracellular BKV load at 34
72 hpi. BKV large T-antigen mRNA and protein expression was unaffected at 24 hpi, but 35
intracellular BKV genome was reduced by 90% at 48 hpi. Late gene expression was 36
reduced by 70 and 90% at 48 and 72 hpi, respectively. Comparing CMX001 or cidofovir 37
equivalent to the EC90 from 24 to 96 hpi demonstrated that CMX001 had a more rapid and 38
enduring effect on BKV DNA and infectious progeny at 96 hpi. CMX001 at 0.31 µM had little 39
effect on overall cell metabolism, but reduced BrdU incorporation and host cell proliferation 40
by 20-30%, while BKV infection increased cell proliferation in both rapidly dividing and near-41
confluent cultures. We conclude that CMX001 inhibits BKV replication with a longer lasting 42
effect than cidofovir at 400x lower levels, with fewer side effects on relevant host cells in 43
vitro. 44
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INTRODUCTION 45
Polyomavirus BK (BKV) infects the majority of humans during childhood and subsequently 46
persists in the renourinary tract with intermittent periods of asymptomatic shedding into urine 47
(8, 24)(Egli et al., 2008;Hirsch & Steiger, 2003). Organ invasive polyomavirus disease is rare 48
and virtually limited to individuals with profound immune dysfunction (17). The prototypic 49
BKV diseases are polyomavirus-associated nephropathy (PyVAN) affecting 1-10% of kidney 50
transplant patients and polyomavirus-associated hemorrhagic cystitis affecting 5-15% of 51
allogenic hematopoietic stem cell transplant recipients (13). Unfortunately, antiviral drugs 52
with specific activity against polyomavirus replication are lacking. Polyomaviruses only 53
encode a few proteins and utilize many host proteins for replication, including the cellular 54
DNA polymerase. In the circular double-stranded DNA genome of approximately 5 kb, the 55
viral early genes encoding the regulatory proteins large- (LT-ag), and small T-antigen (sT-56
ag) are separated by the non-coding control region (NCCR) from the late genes encoding 57
the capsid proteins VP1, VP2 and VP3 as well as agnoprotein. Cell culture and biopsy 58
studies indicate that the BKV replication cycle takes approximately 48h to 72h for 59
completion in renal tubular epithelial cells (1, 21, 33) where early gene expression starts 12-60
24 hours post-infection (hpi), followed by the bi-directional replication of the episomal DNA 61
genome at 36 hpi and late gene expression thereafter (5). The replication rate is accelerated 62
for BKV variants derived from kidney transplant patients with rearranged NCCR (11, 22). 63
64
The most successful strategy to prevent or treat polyomavirus-associated nephropathy in 65
kidney transplant recipients is currently the reduction of immunosuppression (15, 26) which 66
is followed by increases of BKV-specific cellular immune responses (4, 6). Despite 67
promising long-term results in some studies (12), approximately 10% of patients may 68
experience subsequent acute allograft rejection. Without intervention, progression to 69
premature graft failure and loss is observed in more than 80% of cases (3, 25, 28). For 70
polyomavirus-associated hemorrhagic cystitis, reducing immunosuppression is perceived as 71
a difficult undertaking since the pathogenesis entails features of an immune reconstitution 72
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disease and often coincides with significant graft-versus-host disease requiring 73
immunosuppressive therapy (7, 14). Some potentially active drugs like cidofovir (CDV), an 74
acyclic nucleotide phosphonate analogue of deoxycytidine mono phosphate (dCMP), have 75
been sporadically used for the treatment of BKV replication (19, 20, 31). However, since 76
immunosuppression usually is eased simultaneously with the CDV treatment it remains 77
unclear whether or not the partly favourable outcomes could be attributed to the anti-78
polyomavirus activity of the drug or recovery of polyomavirus-specific immunity. 79
80
In vitro studies have shown an effect of CDV on BKV replication in human embryonic lung 81
fibroblast cells (WI-38) (9) and in primary human renal tubular epithelial cells (RPTECs) (1). 82
In RPTECs, CDV inhibited BKV replication in a dose-dependent manner with a 90% 83
effective concentration (EC90) at 40 µg/mL (143 µM) (1). The inhibition was mediated at the 84
step of BKV DNA replication, but also decreased both host cellular DNA replication and 85
metabolic activity as correlates of nephrotoxicity in vivo. Another caveat of CDV is that it 86
must be given intravenous and the patients therefore need to be hospitalized. Recently a 1-87
O-hexadecyl-oxypropyl lipid conjugate of CDV (HDP-CDV) denoted CMX001 became 88
available. Unlike CDV, the conjugate seems to be taken up by cells in a manner similar to 89
lysophosphatidylcholine followed by liberation of CDV by phospholipase cleavage and 90
subsequent anabolism of CDV to the active diphosphate antiviral, CDV-PP. Studies of single 91
and repeated dosing in animals and in human volunteers or patients ranging from 0.1 mg/kg 92
to 4.0 mg/kg has shown no evidence of nephrotoxicity to date (18). In a previous study, 93
CMX001 has been reported to inhibit BKV replication in human fetal fibroblasts, but the 94
mechanistic details were not reported (27). Here we report on the effects of CMX001 on 95
BKV replication in RPTECs which are the primary target of BKV in PyVAN. 96
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MATERIALS AND METHODS 97
Cells and virus 98
Primary human renal proximal tubule epithelial cells (RPTECs) (Lonza, 99
www.lonzabioscience.com) were propagated as described by the manufacturer. No latent 100
BKV could be detected by PCR of intracellular DNA. All experiments were performed with 101
RPTECs at passage 4 and BKV-Dunlop supernatants and gradient-purified virus from Vero 102
cells. In addition to being among the most authentic cells for in vitro studies on BKV, 103
RPTECs are about 10 times more permissive for BKV than Vero cells. 104
105
Infection and CMX001 treatment 106
Before each experiment, CMX001-060 from here on called CMX001, was freshly dissolved to 107
1 mg/ml in methanol/water/ammonium hydroxide (50/50/2) and further diluted in RPTEC 108
growth medium. About 50% confluent RPTECs were infected with BKV-Dunlop MOI 1 for 2 h 109
before removing infectious units by washing and replacing the growth medium with or without 110
CMX001 unless indicated otherwise. Non-confluent dividing cells were chosen in order to get 111
a high level BKV replication. The MOI was determined by a modified plaque assay by serial 112
dilution and seeding of gradient-purified virus or BKV supernatants onto RPTECs. Three 113
days post infection cells were fixed and stained for agnoprotein or VP1 and the number of 114
infectious virus in the inoculums calculated. 115
116
Cytotoxicity and Cell Proliferation Assay 117
The mitocondrial metabolic activity was monitored by the colorimetric WST-1 assay (Roche, 118
Rotkreuz, Switzerland) measuring reduction of the Tetrazolium salt, WST-1, by 119
mitochondrial dehydrogenases. DNA synthesis was quantified by colorimetric measurement 120
of BrdU incorporation into DNA using the “Cell proliferation ELISA, BrdU” kit (Roche). The 121
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results were analysed by the Excel XL fit program for curve fitting to determine the EC90 and 122
the 90% cytotoxic concentration (CC90). The attachment and proliferation of the cells was 123
measured as impedance using E-plates and the xCelligence system (Roche). In order for 124
the RPTECs to attach and proliferate on the E-plates, the plates were first coated with 125
fibronectin. Next the background impedance of the plates was monitored by addition of 100 126
µl medium to each well, before the plate was connected to the system and checked in the 127
cell culture incubator for proper electrical-contacts. Subsequently, 100 µl cell suspension 128
containing the indicated cell numbers was seeded. To determine the effect of BKV infection 129
and CMX001 treatment, about 24h after seeding 150 µl of the media was replaced with 130
fresh media with or without purified BKV-Dunlop in the presence or absence of CMX001 131
(final concentration of 0.31 µM). The cells were grown for 96h and impedance was 132
measured every 15 minutes for the first 6h then every 30 minutes. Impedance was 133
expressed as an arbitrary unit called the Cell Index. 134
135
RNA extraction and cDNA synthesis 136
Expression levels of mRNA was quantified by reverse transcription quantitative PCR (RT-137
qPCR) as described previously (1). At 24, 48, and 72 hpi cells were lysed and total RNA 138
extracted using the mirVana PARIS kit (Ambion). RNA samples were treated with DNase 139
turbo (Ambion) to remove residual DNA before the RNA quality was checked by agarose gel 140
electrophoresis and RNA concentration was determined using the nanodrop method. cDNA 141
was generated from 250 ng RNA per sample using the High Capacity cDNA kit (Applied 142
Biosystems). 143
144
DNA extraction 145
To assay extracellular BKV loads, cell culture supernatants were harvested at 24, 48, and 146
72 hpi and frozen at -70°C until automatic extraction by a robot (GenoM-48, Qiagen, 147
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www.qiagen.com). For intracellular BKV loads, cells were washed, trypsinized and pelleted 148
at 220g for 10 min, resuspended in G2 buffer from the MagAttract DNA Mini M48 kit 149
(Qiagen) and frozen at -70°C until extraction with the same robot. 150
151
Quantitative PCR for BKV DNA and cellular gene detection 152
To quantify intracellular or extracellular BKV DNA load, qPCR with primers and probe 153
targeting the large T-antigen (LT-ag) gene was used (16). For normalization of intracellular 154
BKV DNA each sample was analyzed in parallel by the qPCR for the gene for 155
aspartoacylase (ACY) to correct for cellular DNA (1, 29). 156
157
Western Blotting 158
Cells were lysed in Cell Disruption buffer (mirVana PARIS kit, Ambion) 24, 48, and 72 hpi 159
and stored at -70°C until separation with SDS–polyacrylamide gel electrophoresis (SDS-160
PAGE) followed by blotting onto PVDF membrane. Detection of BKV and cellular proteins 161
was performed with polyclonal rabbit antiserum directed against LT-ag (1:2000), VP1 162
(1:10000), or agno (1:10000) (1, 32) and a monoclonal mouse antibody directed against 163
GAPDH (Ab8245; 1:5000, Abcam, www.abcam.com) followed by anti-rabbit and anti-mouse 164
infrared dye-labeled secondary antibodies (IR Dye 800, Rockland, www.rockland-inc.com 165
and Alexa Fluor 680, Invitrogen, www.invitrogen.com) both 1:5000 before detection and 166
quantification using the Licor Odyssey and the corresponding commercially available 167
software program (www.licor.com/bio/QuantitativeWesterns). The fluorescent signals are 168
considered to be stable with a greater dynamic range than ECL chemiluminescence. In brief, 169
the protein band signals were measured as integrated intensity (pixel-mm2) using the median 170
lane background method with top/bottom segment of border width of 1. Expression of viral 171
proteins was normalized against expression of cellular GAPDH protein on the same blot 172
labelled with a different color whereby the results in untreated cells at each time point were 173
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set as 100%. 174
175
Immunofluorescence staining, microscopy and digital image processing 176
Cells were washed in PBS, fixed in 100% methanol for 10 min and blocked with 3% goat-177
serum in PBS for 30 min both at room temperature, followed by incubation with primary and 178
secondary antibodies for 30 min at 37°C and at room temperature, respectively. Primary 179
antibodies were monoclonal anti-SV40 LT-ag antibody (Pab416; 1:100, Chemicon, 180
www.chemicon.com) and polyclonal rabbit antiserum directed against agno or VP1 (both 181
1:1000). The secondary antibodies were anti-mouse conjugated with AlexaFluor 568 and 182
anti-rabbit conjugated with AlexaFluor 488 (1:500; Molecular Probes, www.invitrogen.com). 183
Nuclei were labeled with DRAQ5TM (Biostatus, www.biostatus.com). Images were collected 184
using a Nikon TE2000 microscope equipped and processed with NIS Elements Basic 185
Research software version 2.2 (Nikon Corporation). 186
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RESULTS 187
Determination of CMX001 effective concentration 90% (EC90) 188
To investigate the effect of CMX001 on BKV progeny, increasing concentrations of CMX001 189
were added 2 hpi and supernatants harvested at 72 hpi. We observed that CMX001 reduced 190
the extracellular BKV load in a concentration dependent manner (Figure 1 a). Recent results 191
from mathematical modeling of BKV replication in kidney transplant recipients suggest that a 192
more than 80% reduction of renal BKV replication must be maintained for up to 10 weeks to 193
observe clearing of plasma and urine viral loads (10). We therefore focussed on the 194
concentration of CMX001 that reduced the extracellular BKV load by 90%. The CMX001 195
concentration of 0.31 µM consistently provided this level of inhibition (Figure 1 a) and was 196
used for further characterization. Immunofluorescence staining of BKV-infected RPTECs 72 197
hpi demonstrated a concentration dependent decrease in number and intensity of large T-198
antigen (LT-ag) and agno expressing cells (Figure 1 b). At concentrations as high as 10 µM 199
CMX001, no BKV-infected cell was observed, but the total cell number was also reduced 200
(see also below). We concluded that CMX001 reduced the expression of early and late BKV 201
proteins and the production of extracellular progeny, but also seemed to have a 202
concentration-dependent effect on the proliferation of RPTECs. 203
204
CMX001 and BKV early gene expression 205
To study the effect of CMX001 at 0.31 µM on BKV early gene expression, we compared LT-206
ag mRNA levels in treated and untreated RPTECS at 24, 48, and 72 hpi by quantitative 207
reverse transcription PCR (RT-qPCR). The results were normalized to the housekeeping 208
gene huHPRT and presented as the changes relative to the untreated sample at 24 hpi. We 209
found no difference in early gene expression at 24 hpi, but a reduction of 33% and 64% 210
were seen at 48 hpi and 72 hpi, respectively (Figure 2 a). Analyzing LT-ag expression by 211
western blotting and normalization to the constitutively expressed enzyme GAPDH revealed 212
a corresponding result showing little difference at 24 hpi, but a 20% to 30% reduction at the 213
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later time points (Figure 2 b). The band seen below LT-ag is a cellular protein of unknown 214
origin cross-reacting with the polyclonal rabbit antiserum used. We concluded that CMX001 215
did not inhibit BKV early protein expression early in the viral life cycle, but later at 48 and 72 216
hpi. 217
218
CMX001 and BKV genome replication 219
Since BKV episome replication is known to occur around 36 hpi (1, 21, 33), we investigated 220
whether the BKV genome replication was affected by CMX001. Intracellular BKV load at 24, 221
48, and 72 hpi was measured by qPCR and normalized to the cell number using the 222
aspartoacylase (ACY) as a cellular reference gene (30). Compared to untreated RPTECs, 223
CMX001 at 0.31 µM reduced the intracellular BKV load by 94% at 48 h and 91% at 72 hpi 224
(Figure 2 c). Thus, we could identify a significant inhibitory effect of CMX001 on intracellular 225
BKV genome replication. This step is known to require LT-ag function which increases viral 226
late gene expression by two synergistic mechanisms, namely by increasing the DNA 227
templates thereby the gene dose per cell for late gene transcription and by activating 228
transcription from the late promoter (5). 229
230
CMX001 and BKV late gene expression 231
To study the effect of CMX001 on BKV late gene expression, we compared VP1 and agno 232
mRNA levels in CMX001 treated and untreated RPTECS at 24, 48, and 72 hpi by RT-qPCR. 233
Late mRNA levels were normalized to the housekeeping gene huHPRT and presented as 234
the changes relative to the untreated sample at 24 hpi. A 93% and 82% reduction was found 235
at 48 and 72 hpi, respectively (Figure 3 a). By western blotting, we found a decrease of VP1 236
of 85 and 96%, respectively while agno was reduced by 97 and 96% (Figure 3 b). 237
238
To investigate the effects of CMX001 on BKV gene expression at the single-cell level, we 239
performed immunofluorescence for the early LT-ag and the late agno and VP1 at 72 hpi. We 240
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found that the number and the intensity of nuclear LT-ag signals was clearly reduced but the 241
decrease was even more pronounced for the late agno and VP1 expression (Figure 3 c). 242
When immunofluorescence staining was performed also at 48 hpi, LT-ag, agno and VP1 243
expression was even more affected indicating a weak increase in BKV protein expression in 244
the treated cells from 48 to 72 hpi (data not shown). Of note, the nuclear VP1 staining in 245
CMX001 treated cells was weak and dispersed and devoid of the strong VP1 stained 246
inclusions characteristic of untreated cells. Immunofluorescence also revealed some 247
refractory cells in the CMX001 treated culture expressing agno at levels comparable to 248
untreated cells even with CMX001 concentrations up to 2.5 µM (Figure 1 b). We also noted a 249
decrease in nuclear DNA fluorescence with increasing CMX001 concetrations suggesting a 250
decrease in total nuclear DNA consisting of BKV and possibly also host DNA which was 251
further addressed by BrdU incorporation (see below). We concluded that CMX001 252
significantly reduces late protein expression, but also inhibits early protein expression at later 253
time points after BKV genome replication had occurred. 254
255
Kinetics of CMX001 inhibition 256
To examine the kinetics of CMX001 inhibition on BKV replication, RPTECs were treated 257
after the 2 h infection for 22h, 46h, 70h or 94h and BKV loads were determined in the 258
supernatants at 96 hpi. At the indicated times, the supernatant was harvested, the cells 259
were washed once and new complete medium was added. At 96 hpi, BKV loads were 260
measured in the supernatants. As shown, CMX001 treatment at 0.31 µM for 22h was 261
enough to reduce the BKV load at 96 hpi by approximately 90% (Figure 4 a). Longer 262
exposure times had only a marginal effect. Under these conditions, CDV treatment at 143 263
µM (40 µg/mL) (1) for at least 46h was needed to reduce the extracellular BKV load to this 264
level. To investigate whether or not the observed reductions in BKV load corresponded to a 265
reduced number of infectious units, the supernatant harvested at the different timepoints 266
were 10-fold diluted and seeded on new RPTECs. Three days post-infection cells were fixed 267
and immunofluoresence staining with antibodies against LT-ag and agno performed. The 268
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results demonstrated that little infectious virus was detectable after treating cells with 269
CMX001 for only 22h, while CDV treatment for 70h was needed to obtain a similar result 270
(Figure 4 b). Next, we examined if CMX001 pre-treatment affected BKV replication. RPTECs 271
were treated for 24h and washed before and after 2 h of BKV inoculation and also at 24 hpi. 272
We found that pre-treatment reduced the extracellular viral load at 72 hpi by more than 99% 273
(Figure 4c). Treatment with CMX001 after the 2 h virus inoculation for 24 h also reduced 274
BKV loads at 72 hpi, but not as effectively as pre-treatment. These observations were 275
confirmed by immunofluoresence staining revealing only few agno expressing cells in the 276
pre-treated wells compared to treatment 24 hpi or untreated wells (Figure 4d). We 277
concluded that CMX001 has a more rapid and enduring inhibitory effect than CDV and can 278
prevent the completion of the BKV lifecycle even if administered for only 24h before or after 279
BKV infection. 280
281
Effects of CMX001 on RPTECs 282
Phase contrast microscopy did not reveal any crude signs of impaired host cell viability 283
during the 3 day exposure to CMX001 at 0.31 µM while a certain reduction was seen at 284
concentration around 10 µM. To use more sensitive assays, we investigated host cell DNA 285
replication and metabolic activity using BrdU incorporation and WST-1 assays in uninfected 286
and infected RPTECs. We found that CMX001 reduced both DNA replication and metabolic 287
activity of infected RPTECs in a concentration-dependent manner (Figure 5 a). Of note, 288
CMX001 at 0.31 µM, the EC90 of BKV replication, induced a 25% reduction in BrdU 289
incorporation, but no significantly altered metabolic activity. As shown in Figure 5, at 290
CMX001 concentration of 10 µM, we observed a 50% reduction in WST-1 activity, but BrdU 291
incorporation was reduced by approximately 90%. 292
293
To investigate the influence of CMX001 at 0.31 µM on proliferation of uninfected and BKV 294
infected RPTECs in real time, we measured the impedance in arbitrary cell index units using 295
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the xCelligence system. Cells were at two different densities: one that permitted exponential 296
growth up to 72h (2000 cells/well, bottom) and one at subconfluency entering confluency 297
within the first 24h after seeding (12000 cells/well, top). One day post-seeding, the medium 298
was replaced, and four conditions examined: i) uninfected and untreated, ii) uninfected, but 299
CMX001 treated, iii) BKV-infected, but untreated or iv) BKV-infected and CMX001 treated. 300
The cell index was measured in 30 min intervals up to 96h. The data showed that BKV 301
infection increased cell proliferation in exponentially growing and in subconfluent cell 302
cultures (Figure 5 b). In exponentially growing cells, CMX001 reduced the rate of RPTEC 303
proliferation by approximately 25% in uninfected cells and by approximately 35% in BKV 304
infected cells at 48h postexposure (72h after seeding). In subconfluent cells, CMX001 had 305
only a minimal inhibitory effect on infected and uninfected cells alike. We concluded that 306
CMX001 at EC90 of 0.31 µM had a certain inhibitory effect on RPTEC proliferation which 307
was inversely proportional to cell density, but did not appear to be toxic at this concentration. 308
309
To determine the selectivity index, we modeled the concentration-dependent effect of 310
CMX001 on the increase in supernatant BKV loads as well as on the BrdU incorporation. 311
The results were fitted to an exponential decay function (supplemental figure 1 a) where the 312
EC90 was between 0.16 µM (SD 0.02) and 0.31 µM (SD 0.04). Similarly, the effect of 313
CMX001 on BrdU incorporation was fitted (supplemental figure 2) indicating a 90% cytotoxic 314
concentration (CC90) of 8.86 µM (SD 1.6). The results suggested that the corresponding 315
selectivity index for CMX001 SI90 (CC90/EC90) could be estimated as being between 29 and 316
55. 317
318
319
320
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DISCUSSION 321
Antiviral drugs with higher efficacy and specificity are needed to improve current outcomes 322
of BKV-mediated nephropathy after kidney transplantation and hemorrhagic cystitis after 323
allogenic hematopoietic stem cell transplantation (31). In this study, we characterized the 324
lipid conjugate 1-O-hexadecyloxypropyl-cidofovir (CMX001) regarding inhibition of BKV 325
replication in human primary proximal tubular epithelial cells. Our results demonstrate that 326
CMX001 at 0.31 µM given after infection was sufficient to reduce the extracellular progeny 327
BKV load by 90% at 72 hpi. Our detailed investigation of the BKV life cycle indicated that 328
CMX001 inhibition occurred at the level of BKV genome replication following the initial 329
phase of early gene expression at 24 hpi. Compared to untreated controls, the increase of 330
intracellular BKV genomes was inhibited between 24 and 48 hpi. Moreover, the burst of late 331
gene expression at 48 and 72 hpi was significantly reduced. In untreated cells, late gene 332
expression results from the synergy of LT-ag mediated activation of late gene expression 333
and the increased copy number of replicated viral genomes. Interestingly, CMX001 334
treatment lead to the same alteration of nuclear VP1 architecture as previously described for 335
BKV-infected RPTECs exposed to leflunomide (2). Since both, CMX001 and leflunomide 336
impair efficient BKV genome replication, the data suggest that this altered replication 337
architecture may not be specific to leflunomide, but partly results from slowed-down 338
replication, possibly as a result of low nuclear genome copy number and hence reduced 339
VP1 expression. CMX001 was active at about 400 times lower concentrations than the EC90 340
for CDV in the same test system (CDV EC90 40 µg/ml =143 µM versus CMX001 EC90 0.31 341
µM). The inhibitory activity of the CMX001 was more immediate and enduring compared to 342
the CDV, requiring an exposure time of only 1 day as compared to 2-3 days for an EC90 of 343
BKV progeny loads at 96 hpi and could be conferred by a pretreatment of 24 h prior to 344
infection. This difference in inhibitory kinetics was also apparent in infectious units when 345
seeding diluted supernatants onto new RPTECs and is likely to result from higher 346
intracellular levels of active antiviral resulting from more rapid uptake of the lipid conjugate 347
(23). When CMX001 was added at 24h before infection a more than 99% inhibition in 348
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extracellular BKV DNA was found. Taken together, the data indicate a significantly 349
enhanced BKV-inhibitory potency of CMX001 over the parent compound CDV. 350
351
Randhawa and colleagues reported increased potency of lipid derivatives of CDV on BKV 352
replication in lung embryonic fibroblasts using the BKV Gardner strain (27). We have used 353
the BKV-Dunlop strain which is characterized by a rearranged NCCR and rapid replication 354
kinetics similar to highly pathogenic variants found in kidney transplant patients (11, 22). 355
Given the mechanism of CMX inhibition at the level of genome replication, it is no surprise 356
that less well replicating non-rearranged BKV are also inhibited as is the polyomavirus JCV 357
(our unpublished data). Our previous work on CDV indicated that the inhibitory activity of 358
CDV was closely linked to inhibitory effects of the host cells: CDV EC90 reduced the 359
proliferation of RPTEC by 30%-40% according to BrdU incorporation, while the overall 360
metabolic activity was reduced by 20% to 30% (1). Given the increased potency of CMX001 361
on BKV replication, it was of considerable interest to monitor effects on the host cells. Our 362
results indicated that CMX001 EC90 had only little effect on the overall metabolic activity of 363
BKV-infected RPTECs and reduced the overall proliferative activity by up to 25%. As 364
reported previously (1), BKV infection by itself increases the metabolic activity of RPTECs 365
over uninfected cells and also increases the proliferative activity as measured by BrdU 366
incorporation. Comparing RPTEC proliferation in a novel real-time proliferation assay, the 367
stimulating effect of BKV infection on cell proliferation was clearly demonstrated on 368
exponentially growing as well as on cell cultures reaching confluency. In accordance with 369
the BrdU results, this assay showed that CMX001 BKV EC90 reduced the proliferation rate of 370
exponentially growing RPTECs by approximately 25%. This effect was less apparent at 371
higher cell densities suggesting that the specificity of CMX001 on BKV infection increased 372
when confluent cells are infected. It can be envisaged that this feature might also contribute 373
to an overall reduced nephrotoxicity especially when treating focal diseases such as 374
polyomavirus-associated nephropathy. There was no discernable effect of CMX001 0.31 µM 375
on early gene expression at 24 hpi, but we consistently observed an approximately 25% 376
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reduction in LTag expression at 48 hpi and 72 hpi, i.e. at a time point after viral genome 377
replication. Since LTag is known to activate the proliferative state of the host cell and the 378
recruitment of building blocks for virus replication, it is possible that CMX001 mediated 379
inhibition of viral genome replication reduced LTag expression at 48 hpi due to a limited 380
gene dosage effect and contributed to the slight decrease in overall proliferative activity at 381
CMX001 of 0.31 µM. Higher concentrations of CMX of up to 10 µM were needed for a 90% 382
effect (see below). CMX001 pretreatment for 24h was also effective to reduce BKV progeny 383
loads, while more than 48 h were required for CDV. Although the reasons for the more 384
pronounced effect of CMX001 over CDV is not clear, we suspect that more rapid uptake 385
and/or equilibration with intracellular nucleotide pools may be involved which might 386
contribute to an effective antiviral state without excessive toxicity. 387
388
CMX001 was found to inhibit BKV replication in human embryonic lung fibroblasts cells (WI-389
38) with a more than 800-fold increased effective concentration-50 (EC50) of 0.13 µM 390
compared to the 115.1 µM observed for CDV (27). These results were obtained by 391
determining the intracellular BKV levels of cells harvested 7 days after infection and 392
normalising to the host cell load using a house keeping gene for the cytotoxic concentration-393
50 (CC50) and indicated a selectivity index-50 (SI50) of 113. Our results aimed at determining 394
the EC90 parameters for the 72 h BKV life-cycle in RPTECs based on a detailed infection 395
model of polyomavirus-associated nephropathy in kidney transplants where these 396
parameters have indicated that an EC90 is needed for contraction of the virus pool and 397
hence clearance of viremia and viruria, by 3 and 10 weeks, respectively (10). Since BrdU 398
incorporation at CMX001 concentration of 10 µM was reduced to approximately 10% of what 399
was found in untreated infected cells as CC90 we could estimate the SI90 as being 32.3. This 400
SI90 must be regarded as very favourable for pre-clinical and clinical studies and extend 401
earlier observations to a clinical relevant host cell model of primary human tubular epithelial 402
cells. 403
404
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Taken together, we conclude that CMX001 like CDV inhibits BKV replication in primary 405
human RPTECs downstream of initial LT-ag expression at the level of viral genome 406
replication. Although polyomavirus replication is dependent on host cell DNA polymerase 407
function, the specificity of CMX001 for BKV replication may result from an enhanced 408
susceptibility of infected cells through their LT-ag mediated activation and preferential 409
recruitment of the host cell replication machinery to the site of viral genome replication. The 410
lipid modification causes a more rapid and enduring antiviral effect of CMX001 at 411
approximately a 400-fold lower concentration than for CDV and an estimated SI90 of 62.5. 412
Together with the oral bioavailability and the lack of nephrotoxicity from CMX001 observed to 413
date in pre-clinical and clinical studies (18), our results suggest that the antiviral potential of 414
CMX001 should be further explored in clinical studies of BKV-mediated diseases. 415
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Acknowledgements 416
We wish to thank Bettina Aasnæs at the Department of Microbiology and Infection Control at 417
the University Hospital of North Norway for excellent technical assistance, Dr Severine 418
Louvel, InPheno AG Basel, Switzerland, for helping with fitting the EC90 and CC90 results. 419
This work was supported by an institutional grant of the University of Basel to HHH, by 420
Extrafunds from the Norwegian Foundation for Health and Rehabilitation to EB and by an 421
unrestricted research grant from Chimerix Inc (USA) to HHH. 422
423
Disclosure statement The authors declare that there is no conflict of interest regarding the 424
role of the sponsors for this research. 425
426
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Figure legends 527
Figure 1: 528
Effect of increasing concentrations of CMX001 on BKV load and expression of BKV 529
proteins. 530
a) RPTEC supernatants were harvested 72 hpi i.e. 70 h post start of treatment with indicated 531
CMX001 concentrations and BKV load was measured by qPCR. DNA load in untreated cells 532
(1.19E+09 Geq/ml) was set as 100%. Determinations were in triplicate. The mean values are 533
shown, and the error bars represent standard deviations. 534
535
b) Indirect immunofluorescence of BKV-infected RPTECs either untreated or treated with 536
indicated CMX001 concentrations. The cells were methanol fixed 72 hpi and stained using 537
as primary antibodies polyclonal rabbit anti-agno serum (green) for visualization of the late 538
agno and the SV40 LT-ag monoclonal Pab416 for visualization of early LT-ag (red). Cell 539
nuclei (blue) were stained with Drac 5.The pictures are taken with the 10x objective. 540
541
542
Figure 2: Influence of CMX001 at 0.31 µM on the BKV-Dunlop early expression and 543
DNA replication in RPTECs 544
a) Early mRNA expression. RNA was extracted from CMX001-treated and untreated BKV-545
infected RPTECs at indicated timepoints. LT-ag mRNA expression was measured by RT-546
qPCR and normalized to huHPRT transcripts. Results are presented as changes in the LT-547
ag mRNA level, with the level in the untreated sample at 24 h p.i arbitrarily set to 1. 548
Determinations were in triplicate. The mean values are shown, and the error bars represent 549
standard deviations. 550
551
b) Early protein expression. Cell extracts from CMX001-treated (+) and untreated (-) BKV-552
infected RPTECs were harvested 24, 48, and 72 hpi and western blot performed with 553
polyclonal rabbit anti-LT-ag serum and with a monoclonal antibody directed against the 554
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housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The anti-LT-555
ag serum also recognizes a cellular protein of unknown origin. 556
c) BKV DNA replication. CMX001-treated and untreated BKV-infected RPTECs were 557
harvested at indicated timepoints and DNA extracted. Intracellular BKV DNA load was 558
measured by qPCR and normalized for cellular DNA using the aspartoacyclase (ACY) qPCR. 559
Data are presented as log Geq/cell. Determinations were in triplicate. The mean values are 560
shown, and the error bars represent standard deviations. 561
562
563
564
Figure 3: Influence of CMX001 at 0.31 µM on the BKV-Dunlop late expression in 565
RPTECs 566
a) Late mRNA expression. RNA was extracted from CMX001-treated and untreated BKV-567
infected RPTECS at indicated timepoints. VP1 mRNA expression was measured by RT-568
qPCR and normalized to huHPRT transcripts. Results are presented as changes in the VP1 569
mRNA level, with the level in the untreated sample at 24 hpi arbitrarily set to 1. 570
Determinations were in triplicate. The mean values are shown, and the error bars represent 571
standard deviations. 572
573
b) Late protein expression. Cell extracts from CMX001-treated (+) and untreated (-) BKV-574
infected RPTECs were harvested 24, 48, and 72 hpi and western blot performed with 575
polyclonal rabbit anti-agno and anti-VP1 serum and with the monoclonal antibody anti-576
GAPDH. 577
c) Early and late protein expression. Indirect immunofluorescence of BKV-infected RPTECs 578
either untreated or treated with CMX001. The cells were methanol fixed 72 hpi and stained 579
using as primary antibodies polyclonal rabbit anti-agno serum, or anti-VP1 serum (both 580
green) for visualization of the late agno and VP1 protein, respectively in combination with 581
the SV40 LT-ag monoclonal Pab416 for visualization of early LT-ag (red). Cell nuclei (blue) 582
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were stained with Drac 5. The pictures are taken with the 20x objective. Inserts show 583
selected BKV-infected cells. 584
585
586
Figure 4: Kinetics of CMX001 at 0.31 µM treatment of BKV-infected RPTECs 587
a) Extracellular BKV load. Cells were infected for 2 h, CMX001 was added added for 22h, 588
46h, 70h or 94h, respectively. At the indicated times, supernatant was removed, cells were 589
washed and new medium added. At 96 hpi all supernatants were harvested and qPCR was 590
performed. Data are presented as BKV load in log Geq/ml. Determinations were in triplicate. 591
The mean values are shown, and the error bars represent standard deviations. 592
b) Infection and immunofluorescence. The supernatant collected 96 hpi from the cells 593
described above, where diluted 1:10 and seeded on new RPTEC cells. 72 hpi cells were 594
methanol fixed and immunofluoresence staining with polyclonal rabbit anti-agno serum 595
(green) and the SV40 LT-ag monoclonal Pab416 was performed (red). Cell nuclei (blue) 596
were stained with Drac 5. The pictures are taken with the 10x objective. 597
c) Extracellular BKV load. CMX001 was added 24h before infection where indicated. Before 598
infection, all cells were washed once with complete medium. Then, BKV was added for 2h, 599
removed and cells washed once with medium. Where indicated, previously untreated cells 600
were now treated with CMX001 for 24h. After 24h all supernatants were removed and cells 601
were washed once more before complete medium was given for 48h. Supernatants were 602
harvested 72 hpi and BKV loads measured by qPCR. Determinations were in triplicate. The 603
mean values are shown, and the error bars represent standard deviations. 604
b) 605
d) Indirect immunofluorescence of BKV-infected RPTECs of cells treated as outline in 606
above. The cells were methanol-fixed and stained using as primary antibodies polyclonal 607
rabbit anti-agno serum (green) for visualization of the viral late gene protein agno and with 608
the SV40 LT-ag monoclonal Pab416 for visualization of viral early protein LT-ag (red). Cell 609
nuclei (blue) were stained with Drac 5. The pictures are taken with the 20x objective. 610
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611
612
Figure 5: Influence of CMX001 on DNA replication, metabolic activity, cell adhesion 613
and proliferation of uninfected and BKV-infected RPTECs. 614
a) Cellular DNA replication was examined with a cell proliferation enzyme-linked 615
immunosorbent assay (ELISA) monitoring BrdU incorporation and metabolic activity was 616
examined with cell proliferation reagent WST-1 measuring WST-1 cleavage. Medium with 617
indicated CMX001 concentrations was added 2 hpi and absorbance measured 72 hpi 618
Absorbance for untreated uninfected cells was set as 100%. Determinations were based on 619
measurements of quintuplicates (5 wells). The mean values are shown, and the error bars 620
represent standard deviations. 621
622
b) For a dynamic monitoring of cell adhesion and proliferation of RPTECs the XCELLigence 623
system was used. RPTECs at a density of 2000 cells/well and 12.000 cells/well were 624
seeded on E-plates. Twenty-seven hours post seeding, 150 µl of the media in each well 625
(totally 200 µl) was replaced with fresh media with or without purified BKV-Dunlop (MOI 5) 626
and with or without CMX001 (total concentration of 0.31 µM) and the cells were left until 96 627
h post cell seeding. 628
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0 0.08 0.16 0.31 0.63 1.25 2.50 5.00 10
20
40
60
80
100
CMX001 concentration (µµµµM)
BK
V l
oad
(% o
fu
ntr
eate
dcell
s)
Figure 1
1.25 µM
Untreated 0.31 µM
5.00 µM2.50 µM
0.65 µM
a
b
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40
30
80
90
Earl
yexp
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n(f
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of
un
treate
dcell
s24h
)
UntreatedCMX001
24
70
60
50
20
10
48 72
Hours postinfection
Figure 2
a
b
LT-ag
GAPDH
24 48 72hpi
- + - + - +CMX001
24
Hours postinfection
48 72
3
2
4
5
6Untreated
CMX001
Intr
acell
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rB
KV
lo
ad
(lo
g G
eq
/cell
)c
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3000
2000
Late
exp
ressio
n(f
old
of
un
treate
dcell
s2
4h
)CMX001
24
6000
5000
4000
1000
048 72
Hours postinfection
Untreated
Figure 3
a
b
GAPDH
24 48 72hpi
Agno
VP1
- + - + - +CMX001
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Agno
MergeDNA
LT-ag
CMX001Untreated
c
Agno
DNA
LT-ag
Merge
VP1
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LT-ag
Merge
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LT-ag
Merge
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Figure 4
7
8
9
10
BK
V l
oad
(lo
g G
eq
/ml)
24 48 72
Duration of treatment (Hours)
96
UntreatedCMX001
CDV
a
b Untreated CMX001 CDV
Du
rati
on
of
treatm
en
t(h
ou
rs)
24
48
72
96
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Untreated Pre-treated Post-treated
BK
V l
oad
(lo
g G
eq
/ml)
7
8
9
10
Untreated Pre-treated Post-treated
c
d
CMX001 treatment
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20
40
60
80
100
Ab
so
rban
ce
(% o
fu
ntr
eate
du
nin
fecte
dcell
s)
120
0 0.08 0.16 0.31 0.63 1.25 2.5 5.0 10
CMX001 concentration (µµµµM)
0Uninfected
BrdU
WST-1
Figure 5
Cell
Ind
ex
1.0 -
2.0 -
3.0 -
4.0 -
5.0 -
I 24
I 48
I 72
I 96
Time post cell seeding (Hours)
Uninfected untreated
Uninfected CMX001 0.31µM
Infected untreated
Infected CMX001 0.31µM
12.000
2000
Cells
/ well
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