Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, August 2006, p. 2806-2813, Vol. 50, No. 8
0066-4804/06/$08.00+0 doi:10.1128/AAC.01641-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line
T. Ueno,1 Y. Eizuru,3 H. Katano,2 T. Kurata,2 T. Sata,2 S. Irie,1 and K. Ogawa-Goto1,2*Nippi Research Institute of Biomatrix, Adachi, Tokyo 120-8601, Japan,1 Department of Pathology, National Institute of Infectious Diseases, Shinjuku, Tokyo 162-8640, Japan,2 Division of Persistent & Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan3
Received 27 December 2005/ Returned for modification 2 March 2006/ Accepted 4 May 2006
Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.
* Corresponding author. Mailing address: Nippi Research Institute of Biomatrix, 1-1-1 Senju-Midoricho, Adachi, Tokyo 120-8601, Japan. Phone: 81-3-3888-5237. Fax: 81-3-3870-9631. E-mail: kgoto{at}nippi-inc.co.jp.
Antimicrobial Agents and Chemotherapy, August 2006, p. 2806-2813, Vol. 50, No. 8
0066-4804/06/$08.00+0 doi:10.1128/AAC.01641-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles:
-
Chevillotte, M., Schubert, A., Mertens, T., von Einem, J.
(2009). Fluorescence-Based Assay for Phenotypic Characterization of Human Cytomegalovirus Polymerase Mutations Regarding Drug Susceptibility and Viral Replicative Fitness. Antimicrob. Agents Chemother.
53: 3752-3761
[Abstract] [Full Text] -
Fukui, Y., Shindoh, K., Yamamoto, Y., Koyano, S., Kosugi, I., Yamaguchi, T., Kurane, I., Inoue, N.
(2008). Establishment of a Cell-Based Assay for Screening of Compounds Inhibiting Very Early Events in the Cytomegalovirus Replication Cycle and Characterization of a Compound Identified Using the Assay. Antimicrob. Agents Chemother.
52: 2420-2427
[Abstract] [Full Text]
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»