Phenotyping of cytomegalovirus drug resistance mutations by using recombinant viruses incorporating a reporter gene

Abstract

A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC50). Changes in the IC50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate.

FIG. 1.

Genome map of recombinant CMV strain T2211. This strain was derived in several steps from strain AD169 (Table 1) and incorporates unique restriction sites in the viral pol and UL97 gene regions, as well as a SEAP reporter gene at US6 driven by the CMV major immediate-early promoter.

FIG. 2.

Growth curves of CMV strains AD169 and T2211. Cultures were inoculated at the indicated MOI, and the culture supernatants were sampled at intervals for the amount of infectious virus. Day zero values represent the infectivity of the input virus. In parallel, SEAP activity (RLU) in the supernatant was measured for T2211 cultured at an MOI of 0.03. Each data point and error bar represents the mean ± standard error of the mean of three experiments.

FIG. 3.

Multiplicity of infection versus SEAP activity at 24 h. Cell cultures were inoculated at various multiplicities of infection up to 0.04, and the SEAP activity in culture supernatants was assayed at 24 h postinoculation. Uninoculated cell culture supernatants had a background RLU reading of <25. The data points were fit to a logarithmic curve, giving the correlation coefficient (R²) shown.

FIG. 4.

SEAP growth curves of CMV strain T2211 under GCV, FOS, and CDV. Cell cultures were inoculated at an MO1 of 0.01 and grown under various drug concentrations (A = GCV, B = FOS, and C = CDV). SEAP activity in culture supernatants was assayed daily at 4 to 7 days postinoculation. Each data point and error bar is the mean ± standard error of the mean of eight experiments.