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. 2000 Jun;44(6):1588-97.
doi: 10.1128/AAC.44.6.1588-1597.2000.

Recombinant green fluorescent protein-expressing human cytomegalovirus as a tool for screening antiviral agents

M Marschall  1 , M Freitag, S Weiler, G Sorg, T Stamminger
Affiliations

Recombinant green fluorescent protein-expressing human cytomegalovirus as a tool for screening antiviral agents

M Marschall et al. Antimicrob Agents Chemother. 2000 Jun.

Abstract

A recombinant human cytomegalovirus (AD169-GFP) expressing green fluorescent protein was generated by homologous recombination. Infection of human fibroblast cultures with AD169-GFP virus produced stable and readily detectable amounts of GFP signals which were quantitated by automated fluorometry. Hereby, high levels of sensitivity and reproducibility could be achieved, compared to those with the conventional plaque reduction assay. Antiviral activities were determined for four reference compounds as well as a set of putative novel cytomegalovirus inhibitors. The results obtained were exactly in line with the known characteristics of reference compounds and furthermore revealed distinct antiviral activities of novel in vitro inhibitors. The fluorometric data could be confirmed by GFP-based flow cytometry and fluorescence microscopy. In addition, laboratory virus variants derived from the recombinant AD169-GFP virus provided further possibilities for study of the characteristics of drug resistance. The GFP-based antiviral assay appeared to be very reliable for measuring virus-inhibitory effects in concentration- and time-dependent fashions and might also be adaptable for high-throughput screenings of cytomegalovirus-specific antiviral agents.

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Figures

FIG. 1
FIG. 1
Construction of recombinant HCMV AD169-GFP and demonstration of the genomic rearrangement by Southern blot analysis. (a) Schematic diagram showing the HCMV genome (upper line) with expansion of the US9/US10 region (middle line) used as an insertion point for the gfp-neo cassette (lower line). The components of the cassette are the US9 recombination element (US9), the loxP recombinase recognition site (loxP), the polyadenylation signal from the bovine growth hormone gene [bGH-poly(A)], the neomycin resistance gene (neoR), the thymidine kinase promoter of herpes simplex virus (Ptk), the enhancer from the polyomavirus mutant PY441 (PY441-enh), the simian virus 40 polyadenylation signal [SV40-poly(A)], a humanized version of the jellyfish GFP gene (gfp-h), SV40 late protein gene 16S/19S splice donor and acceptor signals (SV40-SD/SA), the CMV immediate-early enhancer/promoter (Pcmv), and the US10 recombination element (US10). Small dots indicate the locations of PmeI, BamHI, and PacI restriction sites. The probe used for Southern blotting is indicated by a filled bar. (b) Plaque-purified recombinant viruses were used for infection of HFF, and virion DNA was isolated from the culture supernatants. After digestion with BamHI, samples were subjected to electrophoresis and Southern blotting. A US9-specific, [32P]dATP-labeled hybridization probe of 2 kb (see panel a) was used for the detection of wild-type (wt) and recombinant-type (rec) DNA fragments. pHM673, plasmid control showing the recombinant-type BamHI fragment; AD169, control with DNA from parental HCMV AD169; AD169-GFP I through VI, DNAs from clones of recombinant AD169-GFP virus; Mock, DNA from the uninfected control.
FIG. 2
FIG. 2
Parameters of viral growth (a), viral GFP expression (b), and GFP quantification (c and d). (a) HFF were infected with either HCMV AD169 or AD169-GFP at an MOI of 0.05. Culture supernatants were harvested at the time points indicated and frozen at −80°C. Collected samples were thawed and used for titering of infectious virus by the plaque assay method. Mean values and error bars for two experiments are shown. (b) HCMV AD169-GFP was used for infection of HFF in serial inoculum dilutions. Seven days postinfection, cells were assayed for GFP expression by automated fluorometry. Mean values and error bars of double determinations are shown. The dilution at which 50% of maximal GFP synthesis was detectable was noted as the GFP-TCID50. (c) Comparison between the PRA and the GFP-based antiviral assay. HFF were infected with one of the three viruses AD169, AD169-GFP, and AD169-GFP314 in 12-well plates at an MOI of 0.002. GCV was incubated at 2 or 10 μM in the overlays or supernatants, respectively. Eight to 10 days (PRA) and 7 days (GFP-based antiviral assay) postinfection, cells were harvested and used for quantification. All determinations were performed in triplicate, and relative values are given as the percentages of values for control infections without GCV (Virus). (d) The reproducibility of the GFP-based antiviral assay was demonstrated by a series of three independent testings which were started on three consecutive days (Assays 1, 2 and 3); for each measurements were taken 7 days postinfection. HFF were infected with AD169-GFP virus as for panel c or were mock infected (Mock). Infected cells were cultivated in the presence of GCV (10 μM), A77 (20 μM), or no inhibitor (DMSO at 0.02%). Cells were lysed, and all samples were measured by GFP fluorometry. Relative GFP expression is given as percentages of expression for control infections without inhibitor (DMSO), and the mean of each triplet, including error bars, is shown.
FIG. 3
FIG. 3
Screening for HCMV antivirals. HFF were used for infection with AD169-GFP virus (GFP-TCID50, 0.5; MOI, 0.002) and treated with known or putative antivirals. All substances were incubated at 20 μM (a) or 100 μM (b) immediately after infection by addition to the culture medium. Seven days postinfection, cells were assayed for GFP expression by automated fluorometry. Mean values of double determinations are shown. Mock, uninfected; Virus, virus infection alone; DMSO, virus infection in the presence of the solvent DMSO; GCV, FOS, CDV, and A77, virus infection in the presence of the respective antiviral (aqueous solutions of GCV, FOS, and CDV; DMSO solution of A77); #1 to #53, virus infection in the presence of putative antivirals (all in DMSO solution; final DMSO concentrations, 0.04% in panel a and 0.2% in panel b).
FIG. 4
FIG. 4
Concentration-dependent HCMV-inhibitory effects (IC50 and (a and b) IC90) and determination of cytotoxicity (c). HFF were used for infection with AD169-GFP virus (GFP-TCID50, 0.5; MOI, 0.002) and treated with known or putative antivirals in a range of concentrations as indicated. All substances were incubated immediately after infection by addition to the culture medium, and cells were assayed for GFP expression on day 7 postinfection. One hundred percent GFP expression was defined for the infection control (Virus) in panel a or the DMSO-treated infection control (DMSO) in panel b. The final DMSO concentration in panel b was 0.2% for all samples. For the determination of cytotoxic effects, uninfected HFF were treated with each one of a selection of putative antiviral compounds and analyzed by the cytotoxicity assay (c). Cell layers were harvested 7 days postincubation and used for the standard quantification of the LDH content in the viable, residual cell layer. Five selected novel compounds (dissolved in DMSO) and GCV (in aqueous solution) were assayed at the concentrations indicated, and the percentages of LDH activity of residual cell layers (cell viability) were expressed in relation to an untreated control. As a control for solvent effects, cells treated with an equivalent serial dilution of DMSO (0.0016 to 1%) were assayed in parallel. All data in all panels were produced by double determinations, and mean values are shown. For abbreviations, see Fig. 3.
FIG. 5
FIG. 5
Flow cytometry analysis of HCMV replication under treatment with antivirals. HFF were grown on 6-well plates, infected with HCMV AD169-GFP (GFP-TCID50, 2; MOI, 0.008) or with the GCV-resistant HCMV mutant AD169-GFP314, and subsequently cultivated in the presence or absence of inhibitory substances as indicated. (a through d) On days 1 (open curves) and 7 (shaded curves) postinfection, cells were harvested, fixed in solution, and analyzed for GFP fluorescence by flow cytometry. FL1-Height, GFP signal intensity; Events, cell number. (e through k) Counterstaining with PI was performed 7 days postinfection to separate the portion of intact cells with high DNA content from cells with low DNA content and debris. FL1-H, GFP signal intensity; FL2-H, PI signal intensity. All experiments in all panels were performed twice; representative data for one experiment are shown. Mock, uninfected control; Virus, virus infection alone; GCV, DMSO, A77, or 54, virus infection in the presence of 10 μM GCV, 0.2% DMSO, 100 μM A77, or 20 μM compound 54, respectively.
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