ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2010, p. 5004–5011 0066-4804/10/$12.00 doi:10.1128/AAC.00259-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 12
Differentiation between Polymorphisms and Resistance-Associated Mutations in Human Cytomegalovirus DNA Polymerase䌤† Meike Chevillotte,‡ Ina Ersing, Thomas Mertens,* and Jens von Einem Institute of Virology, Ulm University Hospital, D-89081 Ulm, Germany Received 22 February 2010/Returned for modification 1 April 2010/Accepted 15 September 2010
Specific mutations in the human cytomegalovirus (HCMV) DNA polymerase (pUL54) are known to confer resistance against all currently licensed drugs for treatment of HCMV infection and disease. Following the widespread use of antivirals, the occurrence of HCMV drug resistance is constantly increasing. Recently, diagnostic laboratories have started to replace phenotypic drug resistance testing with genotypic resistance testing. However, the reliability and success of genotypic testing highly depend on the availability of high-quality phenotypic resistance data for each individual mutation and for combinations of mutations, with the latter being increasingly found in patients’ HCMV isolates. We performed clonal marker transfer experiments to investigate the impacts of 7 different UL54 point mutations and also of combinations of these mutations on drug susceptibility and viral replicative fitness. We show that several mutations—S695T, A972V, K415R, S291P, and A692V—of suspected but uncertain drug susceptibility phenotype, either alone or in combination, were not relevant to antiviral drug resistance. In contrast, the combination of two mutations individually characterized previously—E756K and D413E—conferred high-grade loss of susceptibility to all three antivirals. Our results have been added to the newly available database of all published HCMV resistance mutations (http://www.informatik.uni-ulm .de/ni/mitarbeiter/HKestler/hcmv/index.html). These data will allow better interpretation of genotypic data and further improve the basis for drug resistance testing. specific mutations in UL54 confer resistance against any or even all three drugs (23). On the other hand, quite a number of mutations found in the two genes have been shown not to confer resistance (7, 11, 12, 19). In order to reliably predict the drug susceptibility on the basis of sequence data, it is indispensable that mutations found in UL97 and UL54 have to be characterized individually or in combination for their quantitative drug susceptibility phenotypes. The favored approach is to perform marker transfer experiments, where the mutation to be investigated is introduced into a sensitive parental virus, which must then be tested for drug susceptibility. Recent studies used bacterial artificial chromosome (BAC) mutagenesis for generation of clonal recombinant HCMV populations and unbiased measurable readouts for quantitative phenotypic testing, such as by the secreted alkaline phosphatase (SEAP) assay (10) and the fluorescence intensity reduction (FIR) assay (5). Only such marker transfer experiments will allow discrimination of polymorphisms, that is, sequence variations that do not reduce drug susceptibility, from actual resistance-conferring mutations. Nevertheless, many mutations found in patients’ HCMV strains still lack such proper characterization (3). Information concerning the effects of combinations of mutations is even scarcer, although such combinations are increasingly found, especially in UL54 sequences from patients’ specimens. The resulting phenotype of combined mutations is difficult to predict, since it has been shown in a few case reports that combined mutations may potentiate resistance (20, 30, 33) or, in contrast, may compensate for and reduce the loss of susceptibility conferred by the single mutations (24). In this study, we performed marker transfer experiments to investigate the impact of combinations of mutations and single
Human cytomegalovirus (HCMV) infections in immunocompetent individuals are mostly asymptomatic, followed by a life-long latent state of infection. However, in individuals with a compromised or immature immune system, such as transplant recipients, AIDS patients, and connatally infected children, HCMV represents a highly relevant opportunistic pathogen (2, 17, 25, 37). Diagnostic monitoring for active HCMV infection and, in many cases, long-term antiviral therapy are essential for patients at risk for severe HCMV disease. Many different factors influence the success of HCMV antiviral therapy: severity and modality of immunosuppression, drug concentrations, and finally, the susceptibility of a patient’s virus population to the administered antiviral drug (3). As a consequence, antiviral drug susceptibility testing may provide relevant data for adjustment of therapy and the clinical outcome. Three systemic antiviral drugs are currently licensed to treat HCMV infections: ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS) (31). All three target the viral DNA polymerase pUL54. Certain mutations in UL97, encoding a viral protein kinase, or in UL54 itself have been associated with resistance against these three drugs. On the one hand, specific mutations in UL97 confer resistance against GCV, whereas
* Corresponding author. Mailing address: Institute of Virology, Ulm University Hospital, Albert-Einstein-Allee 11, 89081 Ulm, Germany. Phone: 49 (0)731 500 65100. Fax: 49 (0)731 500 65102. E-mail: thomas
[email protected]. † Supplemental material for this article may be found at http://aac .asm.org/. ‡ Present address: Robert Koch Institute, D-13353 Berlin, Germany. 䌤 Published ahead of print on 27 September 2010. 5004
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TABLE 1. vTB65g-derived recombinant HCMV strains Recombinant virus
UL54 genotype
Parental virus
vTB65g vS695Tg vA972Vg vS695T-A972Vg vA692Vg vK415Rg vS291Pg vK415R-S291Pg vE756Kg vD413Eg vE756K-D413Eg
wta S695T A972V S695T A972V A692V K415R S291P K415R S291P E756K D413E E756K D413E
TB40-BAC4 vTB65g vTB65g vS695Tg vTB65g vTB65g vTB65g vK415Rg vTB65g vTB65g vE756Kg
a
wt, wild type.
point mutations in HCMV polymerase on drug susceptibility and viral replicative fitness using the FIR assay. All mutations originate from patients with anti-HCMV therapy failure and are previously uncharacterized. S692T and A972V (suspected to confer GCV resistance) and K415R and S291P (found in a FOS-resistant patient isolate) were analyzed in combination and alone. In addition, the single mutation A692V, located just outside the DNA polymerase domain II at a highly conserved position among sensitive isolates, was also tested. Furthermore, two mutations, E756K and D413E, already proven to confer resistance, were combined in one recombinant virus to determine their possible compensatory or additive effects. MATERIALS AND METHODS Generation of recombinant viruses. The recombinant HCMV strains bearing the desired point mutations in UL54 were generated using a markerless two-step RED-GAM BAC mutagenesis protocol (38). Single UL54 mutations were introduced into parental BAC TB65g (derived from parental strain TB40-BAC4 [5]) bearing wild-type UL54 and a pp65-enhanced green fluorescent protein (pp65-EGFP) fusion or a parental BAC already bearing another UL54 mutation and a pp65-EGFP fusion (Table 1). The mutagenesis protocol was performed in Escherichia coli GS1783 as described previously (5). Primers for generation of the initial PCR product contained homologous sequences of HCMV both upstream and downstream of the site of insertion and are homologous to the pEP-EGFP_in template plasmid sequence (see the underlined sequence in Table S1 in the supplemental material). MRC-5 cells were used between passages 22 and 26 for virus reconstitution from BAC DNA as described previously (4). Human foreskin fibroblasts (HFFs) were used between passages 14 and 20 for all following infection experiments. A cell-free virus stock was prepared after propagation of the viruses on HFFs and was titrated for infectivity by enumeration of cells that stained positive at 48 h postinfection for HCMV immediate early 1/2 and UL44 antigens (monoclonal mouse anti-cytomegalovirus, clones CCH2 and DDG9; Dako). The UL54 gene of each stock virus was sequenced prior to the experiments. Phenotypic assays of recombinant viruses. The FIR assay was performed as described previously (5), with the exception that the method of 50% effective concentration (EC50) calculation was slightly changed. Briefly, 1.3 ⫻ 104 HFFs per well of a 96-well plate were seeded 2 days prior to infection at a multiplicity of infection (MOI) of 1. At the same time, FOS, GCV, or CDV was added to the wells in 2-fold serial dilutions. After 3 days, the drug-containing medium was renewed, the cells were fixed with 4% paraformaldehyde at day 5 or day 7 postinfection (5), and the fluorescence signals were measured in a plate spectrofluorometer (Flx-Xenius). The mean of the background fluorescence signals of mock-infected cells was subtracted from all fluorescence signals. These data were fit by nonlinear least-squares curve fitting to a three-parameter Hill equation for one-site inhibition derived from the four-parameter logistic function originally established for the analysis of families of sigmoidal curves (18) using GraphPad Prism, version 5.01, software. The fluorescence signals were first normalized (maximum signals were set at 100%), and the data sets were then
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transformed by the use of x ⫽ log(X) to set the drug concentration, where x is the transformed value of drug concentration and X is the untransformed value of drug concentration. Then, a nonlinear regression using the sigmoidal doseresponse curve Y ⫽ bottom ⫹ (top ⫺ bottom)/(1 ⫹ 10ˆ[(log EC50 ⫺ x)]) was performed, where Y is the relative fluorescence intensity (Fig. 1). The parameter “top” represents the minimum response when no drug is added; in the case of the FIR assay, top is the maximum fluorescence. This parameter was set as a constant shared value for EC50 calculations for all data sets in one experiment. The parameter “bottom” represents the response at the maximum drug concentration; in the case of the FIR assay, bottom is the remaining fluorescence. In contrast to the parameter top, the parameter bottom was calculated individually for each virus and each drug from the fitted sigmoidal curve. Finally, EC50s were derived as the x value corresponding to the Y value halfway between top and bottom. Using this calculation, the obtained EC50 fulfills its definition of expressing the drug concentration that provides half-maximum inhibition. Statistical analysis of the EC50s obtained for the polymerase mutants compared to the EC50 for parental virus strain vTB65g was performed using the Mann-Whitney U test. This test was also used for determination of synergistic effects (meaning a significant increase in the EC50s of more than just the addition of the values) in the case of the vE756K-D413Eg double mutant virus. Fluorescence intensities from infected cells cultivated without antiviral drugs (top values) were used for determination of polymerase activities. In parallel and for confirmation, HCMV genome copy numbers from infected cells were determined by real-time PCR as described previously (5). Infection with equal amounts of virus was controlled by titration of the inocula. Statistical analysis was performed using a one-way analysis of variance (ANOVA) and Bonferroni’s multiple-comparison test. EC50s were confirmed once by plaque reduction assay. Yield reduction assays using viruses vTB65g, vE756Kg, vD413Eg, and vE756K-D413Eg were performed as described elsewhere (6), with slight changes. HFFs were infected at a MOI of 0.01 and incubated with or without 150 M FOS. After 3 days, the drug-containing medium was renewed and the supernatants were harvested at day 5 postinfection and subsequently titrated on HFFs. Yield reduction factors were calculated as the virus titer obtained without FOS incubation divided by the virus titer obtained with FOS incubation from four independent experiments. Viral growth curves obtained by determination of fluorescence intensity were prepared as described previously (5). Briefly, HFFs were cultivated in minimum essential medium (MEM) without phenol red (Gibco/Invitrogen, Karlsruhe, Germany) and infected with the respective viruses at an MOI of 1. The relative fluorescence intensity (in arbitrary units [AU]) of the infected cells was then measured at the indicated time points in a plate spectrofluorometer (FlxXenius). The relative fluorescence intensity of mock-infected HFFs in MEM without phenol red was used as the baseline. In parallel and as a control, the culture supernatants were collected at the indicated time points, pooled, and subsequently titrated on HFFs.
FIG. 1. Exemplary normalized data sets and fitted sigmoidal doseresponse curves for FOS and viruses vTB65g and vE756K. Values for vTB65g are shown as filled circles, and values for vE756Kg are shown as empty squares. Fifty percent inhibition is shown on the y axis, and the EC50 is readable on the x axis for the respective viruses. Solid line, vTB65g; dotted line, vE756Kg. Each data point and error bar represents the mean ⫾ standard deviation of a triplicate determination for one data set.
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Genotypes and EC50s of recombinant HCMV
HCMV strain
vTB65g vS695Tg vA972Vg vS695T-A972Vg vA692Vg vK415Rg vS291Pg vK415R-S291Pg vE756Kg vD413Eg vE756K-D413Eg
HCMV strain UL54 genotypea
wt S695T A972V S695T A972V A692V K415R S291P K415R S291P E756K D413E E756K D413E
EC50 by FIR assay GCV
CDV
FOS
Mean ⫾ SD
Ratiob
n
Mean ⫾ SD
Ratiob
n
Mean ⫾ SD
Ratio
nb
1.10 ⫾ 0.40 1.22 ⫾ 0.13 1.15 ⫾ 0.42 0.93 ⫾ 0.42 1.32 ⫾ 0.42 1.79 ⫾ 0.32 1.32 ⫾ 0.13 0.95 ⫾ 0.36 1.49 ⫾ 0.45 4.40 ⫾ 2.11 4.25 ⫾ 1.10
1.1 1.0 0.8 1.2 1.6 1.2 0.9 1.4 4.0 3.8
24 4 5 5 5 4 4 4 8 8 9
0.18 ⫾ 0.07 0.09 ⫾ 0.02 0.15 ⫾ 0.04 0.18 ⫾ 0.06 0.21 ⫾ 0.04 0.20 ⫾ 0.13 0.14 ⫾ 0.08 0.20 ⫾ 0.08 0.25 ⫾ 0.11 2.22 ⫾ 0.66 3.56 ⫾ 2.18
0.5 0.9 1.0 1.1 1.1 0.8 1.1 1.4 12 20
26 4 4 4 5 4 4 6 11 6 6
41 ⫾ 10 50 ⫾ 12 38 ⫾ 3 43 ⫾ 15 54 ⫾ 5 43 ⫾ 16 22 ⫾ 3 29 ⫾ 10 105 ⫾ 45 35 ⫾ 13 246 ⫾ 133
1.2 0.9 1.0 1.3 1.1 0.5 0.7 2.6 0.8 6.0
22 4 4 4 6 4 5 4 6 8 5
a
All isolates of strain UL97 were wild type (wt). Ratio EC50 for the indicated strain to that for sensitive parental strain vTB65g; ratios of more than 2-fold are shown in boldface type; n, number of independent experiments performed in triplicate. b
Analysis of published quantitative susceptibility phenotypes. From a previously published database of published HCMV resistance mutations (3), information on quantitative drug susceptibility phenotypes (EC50 ratios obtained for polymerase mutations) was collected. Only marker transfer experiment-proven phenotypes were taken into account. EC50 ratios from previous reports were included in a graphic evaluation (5, 6, 8, 9, 11, 13, 14, 15, 16, 19, 21, 26, 27, 30, 33, 35, 36, 39). In addition, the EC50 ratios obtained in this study (Table 2) were entered into the same graph.
RESULTS Marker transfer analysis of HCMV polymerase mutations. In order to investigate the impact of insufficiently characterized HCMV polymerase point mutations on drug susceptibility and viral replicative fitness, we introduced the respective individual mutations (S695T, A972V, A692V, K415R, S291P) and combinations of mutations (S695T-A972V and K415R-S291P) into drug-sensitive parental virus vTB65g. vTB65g is derived from endotheliotropic strain TB40-BAC4 (34) and carries a fusion of EGFP to the C terminus of pp65 (UL83). This reporter strain allows determination of EC50s (FIR assay) and viral replicative fitness by measuring the fluorescence intensities of infected cells (5). All mutations were introduced by markerless BAC mutagenesis, which especially enabled the introduction of the combination of mutations (38). Polymerase mutations S695T and A972V were originally found together in one clinical specimen from a patient treated with GCV and are of undetermined significance for antiviral drug resistance (32). After marker transfer of the single mutations as well as of the combination of both mutations into drug-sensitive parental virus vTB65g (Table 1), none of the reconstituted clonal viruses showed altered susceptibility to GCV, CDV, or FOS using the FIR assay (Table 2). Mutations S291P and K415R, initially found together in one clinical specimen, were previously analyzed in an in vitro DNA polymerase assay. Both mutations have been shown to reduce susceptibility to FOS when they are present in combination but not when each one is present alone (22). In this study, we investigated the impacts of these mutations, alone and in combination, after marker transfer into vTB65g in cell culture. We could not find reduced susceptibility to GCV, CDV, or FOS either for the two single mutants or for the double mutant,
which is partially in contrast to the previously reported result (Table 2). Polymerase mutation A692V had originally been detected in a clinical isolate sensitive to GCV and FOS (29). The same mutation has also been found in an HCMV sequence obtained from a patient treated with GCV and FOS (32). In our recombinant virus vA692Vg, we did not find relevant increases in the EC50s compared to those for the parental vTB65g virus (Table 2). Generation and phenotyping of the two recombinant viruses vE756Kg and vD413Eg using the FIR assay have already been described (5). Polymerase mutation E756K has been shown to confer reduced susceptibility to FOS and only marginal susceptibility to GCV, whereas D413E has been shown to confer reduced susceptibility to the nucleoside analogues GCV and CDV (5, 6). We found a 2.6-fold difference in the FOS EC50s of vE756Kg, and the fitted curves for FOS differed significantly between vTB65g and vE756Kg (Fig. 1). Mutation D413E had previously been associated with slightly increased susceptibility to FOS (6). To test whether this mutation is able to compensate for decreased susceptibility to FOS, we combined the two mutations D413E and E756K, resulting in recombinant virus vE756K-D413Eg (Table 1). Unexpectedly, instead of reduced EC50s, we found a synergistically increased EC50 for FOS in the double mutant (P ⫽ 0.0441 compared to the EC50 for vE756Kg and P ⫽ 0.0016 compared to the EC50 for vD413Eg; Table 2), showing that the D413E mutation does not compensate for the E756K mutation but reduces sensitivity to FOS instead. Also, for CDV, a further increase in EC50s was found in the double mutant compared to the EC50s for the single mutants (P ⫽ 0.0011, and thus statistically significantly different, compared to the EC50 for vE756Kg, but P ⫽ 0.3290, and thus not statistically significantly different, compared to the EC50 for vD413Eg; Table 2). The GCV EC50s for the double mutant were increased but not more than they were for the vD413g single mutant. An increased susceptibility to FOS due to mutation D413E had previously been determined by a yield reduction assay which measures the residual release of infectious virus with a fixed antiviral drug concentration compared to that achieved
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TABLE 3. FOS drug susceptibility of recombinant HCMV HCMV strain
EC50 ratio for FOS
Yield reduction factor a
vTB65g vE756Kg vD413Eg vE756K-D413Eg
1 2.6 0.8 6.0
158 ⫾ 100 3⫾1 105 ⫾ 76 3⫾1
a Yield reduction factors are expressed as means ⫾ standard deviations of 4 assays with 150 M FOS.
with no drug, which is expressed as the yield reduction factor (6). To further analyze sensitivity to FOS, we performed yield reduction assays for recombinant viruses vTB65g, vE756Kg, vD413Eg, and vE756K-D413Eg, as described previously (6), by using 150 M FOS. We found very low yield reduction factors for recombinant viruses vE756Kg and vE756K-D413Eg compared to the yield reduction factor for sensitive wild-type virus vTB65g (Table 3), indicating a loss of susceptibility and confirming our results from the FIR assay. In contrast, yield reduction factors induced by FOS for virus vD413Eg were not either significantly reduced or significantly elevated compared to those for vTB65g, indicating neither a loss of susceptibility nor increased susceptibility. However, these findings are in line with the result from the FIR assay (Table 3). Replicative fitness phenotypes of HCMV polymerase mutants. Another impact of HCMV polymerase mutations, apart from conferring antiviral drug resistance, is modulation of polymerase activity, often resulting in reduced viral replicative fitness (40). As this parameter might also be relevant to the clinical outcome of HCMV infection, we performed growth kinetics analyses using our recombinant viruses. We had shown previously that determination of viral growth was possible by measuring the fluorescence intensities of infected cells over time (5). As a control, the amount of infectious virus in the cell supernatants was determined by titration. We could not find significantly impaired growth for our recombinant viruses vS695Tg, vA972Vg, vS695T-A972Vg, vA692Vg, vK415g, vS291P, and vK415R-S291Pg (data not shown). We also did not find a significantly reduced polymerase activity for these recombinant viruses compared to the polymerase activity of wild-type virus vTB65g (data not
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shown). In contrast, we found impaired growth of recombinant viruses vE756Kg and vD413Eg, as published previously (5), and for the vE756K-D413Eg double mutant virus (Fig. 2A). Growth defects of about half a log in titer (Fig. 2B), together with 2-fold reduced fluorescence intensities in cells infected with vE756Kg, vD413Eg, and vE756K-D413Eg, indicated reduced polymerase activities (Fig. 3A). Finally, we confirmed the reduced polymerase activity by detection of newly synthesized HCMV DNA by real-time PCR (Fig. 3B). DISCUSSION Early detection of emerging virus mutants associated with reduced antiviral drug susceptibility in patients may become a major challenge during long-term antiviral therapy or even prophylaxis. Genotypic drug resistance testing of HCMV is fast, may even allow the detection of resistant virus subpopulations, and is therefore becoming the method of choice. However, its feasibility highly depends on the phenotypic characterization of all potential resistance-associated mutations detected in UL97 and UL54, as well as combinations of mutations found in patients’ HCMV isolates. From a virological point of view but potentially also for clinical use, it may additionally be of interest to determine the viral replicative fitness of the respective mutant viruses. In the present study, we analyzed the effects of several insufficiently characterized UL54 mutations on drug susceptibility and viral replicative fitness using marker transfer by BAC mutagenesis and FIR assay. UL54 mutations S695T and A972V were originally found together in one clinical specimen of a patient with clinical progression of HCMV disease, despite treatment with valganciclovir (32). Although viral resistance was suspected, we could show that the introduction of neither the single mutations nor the combination of both mutations into our drug-sensitive reporter virus resulted in reduced susceptibility. We can conclude that changes at these positions represent polymorphisms of pUL54 and also that the combination of these two mutations is irrelevant to drug susceptibility. This is also supported by data reported for another mutation at amino acid position 972, which also did not lead to reduced susceptibility to GCV, CDV, and FOS (6). Mutations S291P and K415R were found together in a clin-
FIG. 2. Growth curves of recombinant HCMV strains. HFFs were infected with the indicated viruses at an MOI of 1. (A) The relative fluorescence intensity (in AU) of the cells was measured at the indicated time points. Each data point and error bar represents the mean ⫾ standard deviation of at least four independent experiments. (B) In parallel, the culture supernatants from the same four independent experiments obtained on days 3, 5, and 7 were sampled, pooled, and titrated on HFFs. p.i., postinfection.
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FIG. 3. Relative fluorescence intensities (A) and relative HCMV genome copy numbers (determined by real-time PCR) (B) of cells infected with vTB65g, vE756Kg, vD413Eg, and vE756Kg-D413Eg at day 5 postinfection. HFFs were infected with the indicated viruses at an MOI of 1. Each column and error bar represents the mean ⫾ standard deviation for at least four independent experiments. Significance was determined by one-way ANOVA and Bonferroni’s multiplecomparison test. **, P ⬍ 0.01; ***, P ⬍ 0.001.
ical isolate obtained from an AIDS patient who failed FOS therapy. In an in vitro polymerase assay, 16-fold reduced FOS susceptibility was found, but, interestingly, only for virus with the combination of these two mutations (22). We could not confirm this loss of susceptibility to FOS for the double mutant virus. A possible explanation for this could be that the in vitro polymerase assay apparently tends to overestimate the quantitative drug susceptibility. For example, in the same publication, a 60-fold FOS EC50 increase due to UL54 mutation V715M was determined (22). Nevertheless, two other publications using conventional marker transfer into a sensitive virus stated that the identical mutation conferred only a 5.5-fold (1) or 9.5-fold (15) reduced susceptibility to FOS in phenotypic drug susceptibility cell culture experiments. We hypothesize that in the viral context, interactions of the polymerase with other viral proteins may influence the polymerase’s susceptibility to antiviral drugs, and these interactions may affect the readout. Therefore, the quantitative susceptibility phenotypes determined by cell-based assays with recombinant viruses might provide results with greater biological and clinical relevance. Nevertheless, clinical validation of the results of antiviral susceptibility testing and the definition of clinically relevant HCMV resistance remain major challenges that need to be addressed in the future. This is obviously also true for a change in medication triggered by the results of susceptibility testing. It is remarkable that all single mutations and combinations
ANTIMICROB. AGENTS CHEMOTHER.
of mutations that we analyzed in our study proved to be polymorphisms not conferring a loss of susceptibility, although they all originated from patients with clinically suspected resistance. However, the phenotypes determined are in good accordance with previously published results for mutations at the same or nearby amino acid positions (Fig. 4). These results strengthen the view that therapy can failure for reasons other than the presence of a drug-resistant virus, such as the level of immunosuppression, the choice of immunosuppressive drugs, and the drug concentration in vivo (3). Polymorphisms in pUL54 that do not result in changes of antiviral drug susceptibility are relatively common (7), thus complicating evaluation of HCMV sequence data. Identification of these polymorphisms via phenotypic characterization is therefore essential before a change of therapy can be recommended on the basis of results from genotypic testing. This is especially important in view of the severe side effects of the second-line drugs FOS and CDV compared to those that occur with the first-line drug GCV. Many published marker transfer experiments have reported mutations that confer increases of EC50 ratios below 2- or 3-fold (Fig. 4). It remains to be elucidated whether such small EC50 increases are in fact relevant to antiviral therapy in patients. The relevance of multiple mutations in UL97 and UL54 to drug resistance has not been adequately addressed until now, even though they are found in patients’ HCMV isolates. Markerless mutagenesis protocols now enable introduction of such multiple mutations into drug-sensitive HCMV strains for recombinant phenotyping. It has been described that combinations of mutations may act synergistically with regard to drug resistance or may act as compensatory mutations. Such a partial compensation of reduced drug susceptibility has previously been described for a pair of UL97 mutations (24). In our study, we investigated whether the D413E mutation was able to compensate at least partially for the reduced FOS susceptibility conferred by the E756K mutation (5, 6). D413E has previously been shown to confer a loss of susceptibility to both nucleoside analogues but increased susceptibility to FOS (6). We found a marginal increase in susceptibility to FOS for isolates with this mutation using the FIR assay, and that increase was the same as the increase described for isolates with this mutation by using the plaque reduction assay (6). However, we were not able to confirm this by a yield reduction assay. The reason for the discrepancy between our findings and those described in the previous report could be due to the difference in viral backgrounds used in the two studies (Towne and TB40-BAC4) or the higher MOI that we used in our study (5, 6). More importantly, instead of compensation we found synergistically increased EC50s for FOS in the case of the double mutant vE756K-D413Eg. It is an interesting finding that a mutation from the UL54 region, where directly neighboring amino acids are not implicated in reduced susceptibility to FOS (Fig. 4), can cause further reduced drug susceptibility when it is combined with another mutation, in this case, E756K. This finding highlights the complexity of changes in the polymerase resulting in drug resistance and the urgent need for phenotypic characterization of combinations of mutations in order to reliably predict drug resistance phenotypes from sequence data. Finally, the replicative fitness of viruses vE756Kg, vD413Eg,
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FIG. 4. Map of HCMV DNA polymerase polymerase (pol) and exonuclease (exo) regions, including a graph of quantitative resistance phenotypes (expressed as EC50 ratios) from a previously published database. Only marker transfer experiment-proven phenotypes were taken into account. If phenotyping of the same mutation has been published several times, mean EC50 ratios are depicted. Results from Table 2 were also implemented (data points indicated by arrows). Results are for GCV (A), CDV (B), and FOS (C). The loci containing mutations are depicted at the bottom. N, N terminus; C, C terminus.
and vE756K-D413Eg was impaired compared to that of parental virus vTB65g due to the inserted point mutations (Fig. 2). We could show that this was probably caused by the reduced polymerase activities of the mutants (Fig. 3). It is conceivable that, on the one hand, severely reduced viral replicative fitness due to UL54 point mutations may limit the success of the
resistant viral subpopulation. On the other hand, it has recently been shown that increased viral replicative fitness itself represents a mechanism of antiviral drug resistance (28). Thus, this parameter may gain importance in the future for the prognosis of the clinical outcomes of HCMV infections. Our study has identified 5 new polymorphisms (S695T,
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A972V, A692V, K415R, and S291P) in pUL54. Our results have been entered into the database of HCMV resistance mutations and the corresponding phenotypes and are accessible for evaluation of genotypic testing results via the new web-based HCMV drug resistance mutations search tool (http://www.informatik.uni-ulm.de/ni/mitarbeiter/HKestler /hcmv/index.html [3]). In order to improve the phenotypic data in this database, more marker transfer experiments and greater characterization of newly found or insufficiently characterized mutations are needed. In the end, these experiments, together with the database, will be important tools for optimization of HCMV antiviral therapy on the basis of viral resistance profiles. ACKNOWLEDGMENTS This work was supported by the Ulm University Hospital and by the Robert Koch-Institut, Berlin, Germany. Meike Chevillotte is a participant in the International Graduate School in Molecular Medicine Ulm, funded by the excellence initiative of the German federal and state governments. We thank Anke Lu ¨ske, Judith Rauen, and Oliver Gru ¨nvogel for excellent technical assistance. We are grateful to Peter Gierschik from Ulm University, Institute for Pharmacology and Toxicology, for guidance with the EC50 calculations.
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14.
15.
16.
17. 18.
19.
20.
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