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Antimicrobial Agents and Chemotherapy, September 2009, p. 3752-3761, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00165-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Fluorescence-Based Assay for Phenotypic Characterization of Human Cytomegalovirus Polymerase Mutations Regarding Drug Susceptibility and Viral Replicative Fitness{triangledown}

Meike Chevillotte, Axel Schubert, Thomas Mertens,* and Jens von Einem

Institute of Virology, University Hospital Ulm, D-89081 Ulm, Germany

Received 5 February 2009/ Returned for modification 17 March 2009/ Accepted 17 June 2009

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.

* Corresponding author. Mailing address: Institute of Virology, University Hospital Ulm, 89081 Ulm, Germany. Phone: 49 (0)731 500 65100. Fax: 49 (0)731 500 65102. E-mail: thomas.mertens{at}uniklinik-ulm.de

{triangledown} Published ahead of print on 22 June 2009.

Antimicrobial Agents and Chemotherapy, September 2009, p. 3752-3761, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00165-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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