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. 2009 Sep;53(9):3752-61.
doi: 10.1128/AAC.00165-09. Epub 2009 Jun 22.

Fluorescence-based assay for phenotypic characterization of human cytomegalovirus polymerase mutations regarding drug susceptibility and viral replicative fitness

Meike Chevillotte  1 , Axel Schubert, Thomas Mertens, Jens von Einem
Affiliations

Fluorescence-based assay for phenotypic characterization of human cytomegalovirus polymerase mutations regarding drug susceptibility and viral replicative fitness

Meike Chevillotte et al. Antimicrob Agents Chemother. 2009 Sep.

Abstract

One essential prerequisite for genotypic drug susceptibility testing of human cytomegalovirus (HCMV) is the phenotypic characterization of mutations identified in the viral protein kinase gene UL97 and the viral DNA polymerase gene UL54 regarding their quantitative impact on drug susceptibility. We developed a new method for phenotypic characterization of UL54 mutations with regard to polymerase activity, viral replication, and drug susceptibility. To determine the most suitable viral indicator gene, enhanced green fluorescence protein was C-terminally fused to the HCMV early-late protein UL83 (pp65) or the late proteins UL32 (pp150) and UL99 (pp28), resulting in reporter viruses vTB65g, vTB150g, and vTB28g. vTB65g proved to be superior to the other constructs due to its favorable signal-to-noise ratio and was therefore used to establish the optimum conditions for our assay. The UL54 E756K and D413E mutations were introduced into vTB65g by markerless bacterial artificial chromosome mutagenesis, resulting in virus strains vE756Kg and vD413Eg. The drug susceptibility phenotypes of vE756Kg and vD413Eg were comparable to those previously reported. Furthermore, we found a reduced replicative fitness of vE756Kg by measuring fluorescence intensity as well as by conventional virus growth kinetics. Decreased fluorescence signals of vE756Kg- and vD413Eg-infected cells at late times of infection suggested a reduced polymerase activity, which was confirmed by real-time PCR quantification of the newly synthesized viral DNAs. This new fluorescence-based assay is a highly reproducible method for the phenotypic characterization of mutations potentially influencing drug susceptibility, viral replicative fitness, and polymerase activity of HCMV after marker transfer.

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Figures

FIG. 1.
FIG. 1.
Growth kinetics of HCMV strains TB40-BAC4, vTB65g, vTB150g, and vTB28g. HFF were infected at an MOI of 5, culture supernatants were sampled at the indicated time points, and infectivity was determined subsequently on HFF. Day zero values represent the input infectivity. Each point and error bar represent the mean ± standard deviation for four independent experiments. p.i., postinfection.
FIG. 2.
FIG. 2.
(A) Fluorescence microscopy (magnification, ×100) of HFF infected with EGFP-labeled HCMV strains vTB65g, vTB150g, and vTB28g. Shown are representative pictures of cells 5 days after infection at an MOI of 1 with subsequent incubation without or with 800 μM FOS. All pictures were taken with the same exposure time in order to allow comparison of the fluorescence signals. (B) Signal-to-noise comparison for strains vTB65g, vTB150g, and vTB28g. Fluorescence signals of infected cells were measured using Flx-Xenius (Safas), and each bar and error bar represent the mean ± standard deviation for values from six wells.
FIG. 3.
FIG. 3.
Fluorescence growth curves for HCMV strain vTB65g grown with GCV (A), FOS (B), and CDV (C). HFF were infected at an MOI of 1 and grown at various drug concentrations. Day zero values represent the relative fluorescence intensity after a 2-h inoculation period and two subsequent washes of the cells with PBS. The relative fluorescence intensity (AU) of the infected cells was measured at the indicated time points. Each point and error bar represent the mean ± standard deviation for values from six wells. p.i., postinfection.
FIG. 4.
FIG. 4.
Comparison of GCV EC50s of HCMV strain vTB65g on different days postinfection. HFF were infected at an MOI of 1 and grown at various GCV concentrations. Each point represents the value for one well. Shown are means and standard errors for 12 values obtained from two independent experiments.
FIG. 5.
FIG. 5.
MOI dependence of EC50s determined by FIR assay. HFF were infected with vTB65g virus at different FOS concentrations and various MOIs ranging from 0.01 to 12. Relative fluorescence intensities (A) and EC50s (B) were determined on day 5 postinfection.
FIG. 6.
FIG. 6.
Growth curves for HCMV strains vTB65g and vE756Kg. HFF were infected with the indicated viruses at an MOI of 1. The relative fluorescence intensity (AU) of the cells was measured at the indicated time points. p.i., postinfection. (A) In parallel, the culture supernatants were sampled, and triplicate samples were pooled and titrated on HFF. Day zero values represent the fluorescence intensity or infectivity after a 2-h inoculation and two subsequent washes of the cells with PBS. Each point and error bar represent the mean ± standard deviation for three independent experiments. □, vTB65g fluorescence; ▵, vE756Kg fluorescence; ▪, vTB65g titer; ▴, vE756Kg titer (fluorescence is plotted on the left y axis, and titers are plotted on the right y axis). (B) In parallel, cells were harvested at the indicated time points, and quantitative real-time PCR was performed in order to determine relative HCMV genome copy numbers. Each point and error bar represent the mean ± standard deviation for three independent experiments. □, vTB65g fluorescence; ▵, vE756Kg fluorescence; ▪, vTB65g genome copies; ▴, vE756Kg genome copies (fluorescence is plotted on the left y axis, and titers are plotted on the right y axis).
FIG. 7.
FIG. 7.
Western blot analyses of cell lysates from HFF infected with HCMV strains vTB65g and vE756Kg at an MOI of 3. Cells were harvested at the indicated time points.
FIG. 8.
FIG. 8.
(A) Relative fluorescence intensities of vTB65g, vE756Kg, and vD413Eg viruses at day 5 postinfection. HFF were infected with the indicated viruses at an MOI of 1. Each column and error bar represent the mean ± standard deviation for at least four independent experiments. (B) Numbers of genome copies in vTB65g-, vE756Kg-, and vD413Eg-infected cells at day 5 postinfection. HFF were infected at an MOI of 1 and collected at the indicated time points. Equal inputs of virus were controlled by titration of the inocula and determination of genome copy numbers from infected cells at 48 h postinfection. The number of HCMV genome copies was determined by real-time PCR. Each column and error bar represent the mean ± standard deviation for four independent experiments.
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