Resistance of Human Cytomegalovirus to Cyclopropavir Maps to a Base Pair 1 Deletion in the Open Reading Frame of UL97 2
3 Running Title: Resistance of HCMV to Cyclopropavir Maps to UL97 4
5 BRIAN G. GENTRY1#, LAURA E. VOLLMER1, ELLIE D. HALL2, KATHERINE Z. 6
BORYSKO3, JIRI ZEMLICKA4, JEREMY P. KAMIL2, JOHN C. DRACH3 7 8
1 Department of Pharmaceutical, Biomedical and Administrative Sciences, College of Pharmacy 9 and Health Sciences, Drake University, Des Moines, Iowa 50311 ([email protected], 10 [email protected]) 11 2 Department of Microbiology and Immunology, Louisiana State University, Shreveport, 12 Louisiana 71103 ([email protected], [email protected]) 13 3 Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 14 Ann Arbor, Michigan 48109 ([email protected], [email protected]) 15 4 Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, School of 16 Medicine, Wayne State University, Detroit, Michigan, 48201 ([email protected]) 17 #Address Correspondence to: 18 19 Brian Gentry, Ph.D. Email: [email protected] 20 Department of Pharmaceutical, Biomedical, and Phone: (515) 271-2980 21 Administrative Sciences Fax: (515) 271-1867 22 Drake University College of Pharmacy and Health 23 Sciences 24 107 Fitch Hall 25 2507 University Ave. 26 Des Moines, Iowa 50310-4505 27
ABSTRACT 29 Human cytomegalovirus (HCMV) is a widespread pathogen in the human population 30
affecting many immunologically immature and immunocompromised patients, and can result in 31 severe complications such as interstitial pneumonia and mental retardation. Current 32 chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and 33 cidofovir. However, high incidences of adverse effects (neutropenia and nephrotoxicity) are 34 prevalent and limit the use of these drugs. Cyclopropavir (CPV), a guanosine-nucleoside analog, 35 is 10-fold more active against HCMV compared to GCV (EC50’s = 0.46 and 4.1 μM, 36 respectively). We hypothesize that its mechanism of action is similar to that of GCV; 37 phosphorylation to a monophosphate by viral pUL97 protein kinase with further phosphorylation 38 to a triphosphate by endogenous kinases resulting in viral DNA synthesis inhibition. To test this 39 hypothesis, we isolated a CPV resistant virus, sequenced its genome, and discovered that base 40 pair 498 of UL97 was deleted. This mutation caused a frame shift in UL97 resulting in a 41 truncated protein that lacks a kinase domain. To determine if this base pair deletion was 42 responsible for drug resistance, the mutation was engineered into the wild-type viral genome and 43 then subjected to increasing concentrations of CPV. The results demonstrate that the engineered 44 virus was approximately 72-fold more resistant to CPV (EC50 = 25.8 ± 3.1 μM) compared to 45 wild-type virus (EC50 = 0.36 ± 0.11 μM). We conclude therefore that this mutation is sufficient 46 for drug resistance and that pUL97 is involved in the mechanism of action of CPV. 47
INTRODUCTION 49 Human cytomegalovirus (HCMV), a β-herpes virus, is a widespread pathogen affecting a 50
majority of the world’s population [1]. Individuals at risk for symptomatic HCMV disease – 51 which includes interstitial pneumonia, encephalitis, and retinitis – are those with deficiencies in 52 T-cell immunity. In addition, with over 4,000 cases reported each year, HCMV is the most 53 common congenital infection in the U.S. and can result in severe mental retardation, hearing, 54 and/or vision loss [2]. Drugs currently approved by the FDA for prophylaxis or the treatment of 55 systemic HCMV infections are ganciclovir (GCV; Figure 1) and its oral prodrug valganciclovir, 56 foscarnet, and cidofovir [3-5]. GCV is the most common therapy option for the treatment of 57 HCMV infections among patients with impaired immunity, particularly those with advanced 58 HIV/AIDS, those undergoing cancer chemotherapy, and recipients of solid organ and bone 59 marrow transplants [6-8]. GCV, an acyclic analog of the nucleoside guanosine, is converted 60 intracellularly to a monophosphate by a viral protein kinase encoded by the HCMV gene UL97 61 [9, 10]. Upon further phosphorylation by cellular enzymes [11, 12], GCV triphosphate competes 62 with endogenous dGTP for incorporation into progeny viral genomes resulting in pUL54 (viral 63 DNA polymerase) inhibition [13, 14]. Once incorporated, GCV also acts as a chain-terminator 64 further inhibiting viral DNA synthesis [15, 16]. However, long term therapy is typically required 65 due to recurrence of infection and reactivation of latent HCMV upon cessation of therapy. As 66 such, drug resistance and adverse effects, such as neutropenia (up to 30% of patients) and 67 nephrotoxicity (up to 50% of patients), are prevalent and limit the use of these drugs [3, 17-19]. 68 With increased use of immunosuppression for cancer chemotherapy and organ transplantation, 69 there is an increasing need for more effective and less toxic drugs to treat HCMV. 70
Cyclopropavir (CPV; Figure 1), a methylenecyclopropane guanosine nucleoside analog, 71 is currently being explored as a viable chemotherapy option for HCMV due to its high potency 72 and low incidence of adverse effects in vivo [20, 21]. Previous studies have demonstrated that 73 CPV is approximately 10-fold more active in vitro (EC50 = 0.46 μM) compared to GCV (EC50 = 74 4.1 μM) without any observed increase in cytotoxicity [22]. Furthermore, CPV elicits a 2- to 5-75 log reduction in murine cytomegalovirus titers in vivo resulting in reduced mortality in SCID 76 mice [21]. Although the mechanism of action of CPV has been hypothesized in previous studies, 77 the exact mechanism by which CPV elicits an anti-viral effect has not been determined [23]. We 78 have established that both CPV and GCV are phosphorylated to monophosphates by viral pUL97 79 protein kinase [24]. Additional phosphorylation to a triphosphate by endogenous kinases is also 80 similar for CPV and GCV [25]. However, viral DNA polymerase inhibition and incorporation 81 into HCMV is known for GCV [13-16] but has not been established for CPV. Furthermore, the 82 necessity of phosphorylation by pUL97 for antiviral activity is unknown. Thus, the goal of this 83 study is to determine whether the viral protein kinase pUL97 is involved in the mechanism of 84 action of CPV. 85 86
MATERIALS AND METHODS 87 Chemicals. CPV was synthesized in the laboratory of J. Zemlicka as previously 88
described [22]. GCV was kindly provided by Hoffman La Roche (Palo Alto, CA). 89 Cell culture procedures. Human foreskin fibroblasts (HFF) were grown in minimal 90
essential medium with Earle’s salts and 10% fetal bovine serum. They were grown at 37 oC in a 91 humidified atmosphere of 3-5% CO2-97-95% air and were regularly passaged at 1:2 dilutions 92 using conventional procedures with 0.05% trypsin and 0.02% EDTA in HEPES-buffered saline 93 [26]. 94
Viral strain. The HCMV Towne strain was kindly provided by M. F. Stinski, University 95 of Iowa. A bacterial artificial chromosome (BAC)-clone of strain AD169 (AD169rv) was 96 generously provided by U. H. Koszinowski, Ludwig-Maximilans University (Munich, Germany) 97 [27]. 98
HCMV plaque reduction assay. HFF cells were seeded at 85,000 cells per well in a 24-99 well cluster dish and infected three days later with HCMV at 100 PFU per well. Two hours post-100 infection, media containing serial dilutions of drug, a final fetal bovine serum concentration of 101 3%, and 0.5% methylcellulose were added. After incubation at 37 oC for 9-11 days, cell 102 monolayers were stained with crystal violet and plaques were enumerated by light microscopy. 103 The number of plaques observed in the presence of each drug concentration was compared to the 104 number observed in the absence of drug to determine drug effect. The percent inhibition of 105 plaque number was plotted against log10 drug concentration. Fifty percent effective 106 concentrations (EC50) were interpolated from the linear portions of the regression lines. All drug 107 dilutions were tested, at least, in duplicate. 108
Selection of CPV-resistant virus. HFF cells were infected with HCMV at a MOI of 109 0.01 and grown in a 25 cm2 tissue culture flask in the presence of 0.5 μM CPV for 2 weeks. 110 Supernatant progeny virus was then passaged in the presence of first 1.0-, then 2.0-μM 111 concentrations of CPV; the duration for each passage was 2 weeks. The resulting virus was 112 further passaged in 2.0 μM CPV for 3 months, frozen in liquid nitrogen, and the titer was 113 determined. The resulting virus stock was purified using the Klein limiting dilution method [28] 114 and termed 2696r. 115
DNA sequencing. Primers for PCR amplification and sequencing were determined 116 based on the published sequence of the Towne strain of HCMV. After PCR amplification, 117 products were run on a 0.8% agarose gel, extracted, and purified using a QIAquick Gel 118 Extraction Kit (Qiagen, Valencia, CA). Sequencing PCR was done with BigDye Terminator 119 cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) and sequencing 120 reactions were separated and detected with an ABI Prism 310 genetic analyzer. Sequences were 121 aligned and edited using Sequencher software (Genecodes, Ann Arbor, MI). 122
Western blot analysis. HFF cells were infected with wild-type HCMV or 2696r at a 123 MOI of approximately 1.0 PFU/mL and grown in a 75 cm2 tissue culture flask. After 4 days, 124 cells were harvested, lysed using lysis buffer (2% NP40, 0.1 M Tris pH 8.8, 0.1% SDS, 0.3 M 125 NaCl), and protein levels were determined using the DC Protein Assay (BioRad Corp., Hercules, 126 CA). Fifty micrograms of protein per sample was electrophoresed under reducing conditions and 127 blotted electorphoretically to nitrocellulose paper (BioRad Corp., Hercules, CA). Detection of 128 pUL97 was performed using a previously characterized polyclonal antibody developed against 129 full-length pUL97 (a generous gift of Don Coen, Harvard University, Boston, MA) [29] and the 130
SuperSignal West Pico Chemiluminescent Substrate imaging system (Thermo Scientific, 131 Rockford, IL). A monoclonal antibody against human actin was used as a loading control. 132
Marker transfer studies. “En passant” mutagenesis of BACs was performed as 133 described elsewhere [30-32] to remove base pair 498 of the UL97 open reading frame from a 134 bacterial artificial chromosome (BAC) clone of HCMV strain AD169, AD169rv (a generous gift 135 of Ulrich Koszinowski) [27]. All manipulations were performed in Escherichia coli strain 136 GS1783 (a generous gift of Greg Smith, Northwestern University, Chicago, IL). Briefly, an 137 excisable kanamycin resistance marker (I-SceI-aphAI) was amplified from a plasmid template 138 using oligonucleotide primers d_bp498_F and d_bp498_Rv, which had been custom synthesized, 139 and purified by polyacrylamide gel electrophoresis (PAGE) by Integrated DNA Technologies, 140 Inc. (Coralville, IA). d_bp498_F: 141 5'- AAA CTT CGG CCA TGT GGT CGT TCG AGT ACG ATC GCG ACG GGA CGT GAC 142 CAG CG T ACG CCG TAG GGA TAA CAG GGT AAT CGA TTT -3'; d_bp498_Rv: 143 5'- TGC CGC CGG TGA AGA GAG CGC GGC GTA CGC TGG TCA CGT CCC GTC GCG 144 ATC GT A CTC GAA GCC AGT GTT ACA ACC AAT TAA CC -3' 145
The resulting PCR product was gel purified using a NucleoSpin Gel and PCR Clean-up 146 kit (Macherey-Nagel, Inc., Bethlehem, PA), then electroporated into E. coli containing the 147 AD169rv BAC. Integrate colonies were resolved to remove the kanamycin resistance marker. 148 The resulting recombinant BAC, AD169rv d498, was confirmed by restriction enzyme digestion 149 analysis, and by DNA sequencing of the modified region (Genewiz, Inc., South Plainfield, NJ). 150
Virus reconstitution. Infectious virus was reconstituted from BAC DNA as described 151 previously [32] by co-transfection of ~0.7 X 105 human foreskin fibroblast cells in a 24-well 152 cluster plate well (Corning, Inc.) with a total of 1 µg DNA containing 750 ng BAC DNA, 200 ng 153
pp71 expression plasmid pSG5-pp71 [33] (a gift of Robert Kalejta, Univeristy of Wisconsin, 154 Madison, WI) and 50 ng Cre recombinase expression plasmid pCAGGS-nlsCre [34] (a gift of 155 Michael I. Kotlikoff, Cornell University, Ithaca, NY), using 7 µL of Superfect transfection 156 reagent (Qiagen, Inc., Valencia, CA), according the manufacturer’s recommendations. 157
HCMV growth assay. HFF cells were seeded and grown to confluence in a 25 cm2 158 tissue culture flask at 37 oC. Cells were then infected with virus at an M.O.I. of 0.02. Two-159 hours post-infection, media was added to the flask and samples were collected every 24 hours 160 over the course of 10 days (collected samples were stored at -80oC until titer could be 161 determined). Titer of virus for each sample was determined by first plating a 96-well plate with 162 HFF cells grown to confluence. 1:3 serial dilutions of samples (200 μL total volume) were then 163 plated and virus was allowed to grow for 7-9 days at 37 oC. Finally, cell monolayers were 164 stained with crystal violet and plaques were enumerated by light microscopy. The virus titer was 165 determined by the following equation: 5 x 3n x number of plaques, where n is the serial dilution 166 in which the plaques were enumerated. 167 168
RESULTS 169 Initial isolation and characterization of 2696r (CPV resistant HCMV). We 170
hypothesize that the mechanism of action of CPV is multi-phase process: initial phosphorylation 171 by viral pUL97 to a monophosphate followed by phosphorylation by endogenous kinases to a 172 triphosphate resulting in inhibition of viral DNA synthesis. Although we have previously 173 demonstrated that CPV is phosphorylated by pUL97 to a monophosphate (the initial step of our 174 hypothesis) [24], it has not been determined if this enzymatic conversion is necessary for CPV to 175 elicit an antiviral effect. Therefore, to test the first step of our hypothesis, HFF cells infected 176 with a CPV-resistant HCMV (2696r) was subjected to increasing concentrations of drug to 177 confirm resistance (Figure 2, Table 1). As expected, 2696r was approximately 25-fold more 178 resistant to CPV (EC50 = 22.5 ± 3.5 μM) compared to wild-type (Towne strain) virus (EC50 = 179 0.91 ± 0.15 μM). In addition, HFF cells infected with 2696r were subjected to increasing 180 concentrations of GCV to determine if the virus is also resistant to this drug. As shown in Figure 181 2, 2696r is approximately 28-fold more resistant to GCV (EC50 = 41.3 ± 2.8 μM) compared to 182 wild-type virus (EC50 = 1.5 ± 0.16 μM). These results demonstrate that 2696r, which is resistant 183 to CPV, exhibits cross-resistance with GCV. 184
DNA sequencing of 2696r. Since 2696r was resistant to GCV and GCV resistance has 185 been mapped to mutations in UL54 and UL97 [17, 18], the open reading frames of UL54 and 186 UL97 in 2696r were sequenced. The results demonstrated that the open reading from of UL54 in 187 2696r contained no mutations whereas base pair 498 of the 2696r gene UL97 was deleted (Figure 188 3). This deletion results in a codon frame shift and the introduction of a stop codon at base pairs 189 502-504 (corresponds to bp 503-505 of wild-type HCMV UL97). The protein product of this 190 mutated gene would be considerably truncated (167 amino acids versus 707 amino acids for 191
wild-type [Towne strain] pUL97) lacking both an ATP binding site and substrate pocket [10, 35, 192 36]. To confirm that pUL97 is truncated in 2696r when compared to wild-type virus, a Western 193 blot was performed (Figure 4). The results depict a significantly lower molecular weight 194 reactive species (MW = 17.8 kDa) in the 2696r lane that is absent from the wild-type lane. In 195 addition, the reactive species corresponding to pUL97 in the wild-type lane (MW = 78.3 kDa) is 196 absent from the 2696r lane. These results demonstrate that pUL97 is indeed truncated in 2696r. 197
Recombinant HCMV UL97Δ498 exhibits resistance to both CPV and GCV. To 198 confirm that the base pair 498 deletion of 2696r is the mechanism by which this virus resists the 199 antiviral effects of CPV, this mutation was engineered into wild-type virus (HCMV UL97Δ498). 200 Since previous studies have demonstrated that UL97-null mutants have a 1-2 log growth defect 201 that is attributable to the loss of kinase activity [29, 37] and the mutation discovered in 2696r 202 would result in a virus without an active pUL97 kinase, both 2696r and HCMV UL97Δ498 were 203 assayed for replication competency and compared to the growth of wild-type virus. As depicted 204 in Figure 5 and consistent with previous results, 2696r (1.0 ± 0.17 x 105 PFU/mL) and HCMV 205 UL97Δ498 (1.5 ± 0.11 x 105 PFU/mL) exhibited growth defects in excess of one log when 206 compared to wild-type (BAC-derived AD169rv) virus (2.8 ± 0.17 x 106 PFU/mL). 207
Following conformation that the engineered virus replicated (albeit at a slower rate than 208 wild-type virus), HCMV UL97Δ498 infected cells were subjected to increasing concentrations of 209 either CPV or GCV and compared to wild-type virus under the same conditions. The results, 210 depicted in Figure 6 and Table 1, demonstrate that HCMV UL97∆498 was approximately 72-211 fold more resistant to CPV (EC50 = 25.8 ± 3.1 μM) and 14-fold more resistant to GCV (EC50 = 212 28.1 ± 6.6 μM) when compared to wild-type (BAC-derived AD169rv) virus (EC50 = 0.36 ± 0.11 213 and 2.0 ± 0.24 μM, respectively). We, therefore, conclude that the base pair deletion mutation 214
in the open reading frame of the HCMV encoded gene UL97 is sufficient for drug resistance and 215 that the protein product of this gene is involved in the mechanism of action of CPV. 216
DISCUSSION 219 CPV is a known inhibitor of HCMV replication [23]. The hypothesized mechanism by 220 which CPV elicits this effect involves the phosphorylation of CPV to a monophosphate by the 221 viral enzyme pUL97. Our current results demonstrate that a previously uncharacterized mutation 222 in the open reading frame of UL97, which would result in expression of a truncated pUL97 223 lacking an active kinase domain, renders the virus resistant to both CPV and GCV. These results 224 establish that pUL97 is necessary for CPV to elicit an antiviral effect and, taken together with 225 our previous results [24], establish that pUL97 is the enzyme responsible for the initial 226 phosphorylation of CPV to a monophosphate. 227 Previous studies have identified mutations within the HCMV genome that confer 228 resistance of virus to CPV. The first example describes a frame shift mutation in UL27 in a virus 229 with wild-type UL97 developed as a result of exposure to CPV [38]. This mutation, presumably, 230 compensates for the loss of pUL97 enzymatic activity, since CPV exhibits some pUL97 231 inhibitory activity and a mutation described for HCMV resistance to maribavir, a potent pUL97 232 inhibitor, occurs in UL27 [38-40]. Next, a recombinant virus lacking a functional pUL97 kinase 233 is 21-fold more resistance to CPV when compared to wild-type virus [23]. The previously 234 uncharacterized mutation that we presented in this study, which causes a frameshift with a 235 concomitant premature translational termination of the viral kinase pUL97 and, presumably, a 236 complete loss of enzymatic function, behaves similarly to the previously characterized UL97-null 237 with regards to growth characteristics (Figure 5) and CPV resistance (Figure 6). Finally, there 238 are mutations that have been generated as a result of exposure to CPV that confer resistance to 239 the drug without the complete loss of enzymatic function [41-43]. These mutations not only 240 confer resistance to CPV, but to GCV as well. The mutation described herein (UL97Δ498) also 241
confers cross-resistance with GCV since GCV requires initial phosphorylation by pUL97 as part 242 of its mechanism of action [9]. 243 Although there are many mutations that confer cross-resistance between GCV and CPV, 244 including the mutation we present above, there are several mutations that confer resistance to 245 GCV, but not to CPV [41, 44, 45]. This suggests that these two drugs either interact differently 246 or occupy different sites, albeit close, within the substrate binding pocket of the pUL97 kinase. 247 We therefore cannot rule out the possibility that certain mutations in UL97 may confer resistance 248 to CPV without conferring resistance to GCV. In addition, mutations that confer HCMV 249 resistance to maribavir [46], a potent pUL97 inhibitor [47], demonstrate no cross-resistance with 250 GCV [48]. This suggests that these two nucleoside analogs either interact differently or occupy 251 distinct sites within the substrate binding pocket of pUL97. Given that CPV demonstrates 252 characteristics of both GCV (substrate of pUL97 [24]) and maribavir (inhibitor of pUL97 [38]), 253 we hypothesize that CPV binds to a site central to these two nucleosides analogs, occupying 254 space that would otherwise be occupied by either GCV or maribavir. We further speculate that, 255 given the possibility that CPV and maribavir occupy a similar site within the substrate binding 256 pocket of pUL97, there could be mutations that confer resistance to CPV and maribavir. Given 257 that maribavir and CPV are currently being evaluated as candidates for clinical trials, an 258 examination of mutations that could confer cross-resistance to both of these nucleoside analogs 259 is warranted. 260 The use of CPV for the treatment or prophylaxis of HCMV disease is promising. Our 261 current results demonstrate that pUL97 is an integral part of the mechanism of action of CPV. 262 As such, it is possible that cross-resistance between CPV and currently approved 263 chemotherapeutics for the treatment of HCMV may occur. However, this has not been observed 264
in clinic. In fact, clinical strains of HCMV resistant to GCV were sensitive to earlier analogs of 265 CPV [49]. Also, increased antiviral activity without any observed increase in toxicity [22] and 266 the ability to achieve therapeutic concentrations in vivo without prodrug modification [5, 20] are 267 a few reasons why CPV should be superior to GCV for the treatment of HCMV disease. Further 268 examination into the mechanism of action and pre-clinical development of this compound is 269 warranted. 270
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