Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Abstract

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.

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

“…Unlike other assays, including PRA, which are based on measuring the spread of progeny virus in cell culture (11,18,22,23,35), our FIR assay allows the determination of EC 50 s from single-cycle virus replication. This accelerates testing and allows indirect evaluation of polymerase activity at higher MOIs.…”

“…Many efforts have been made to develop new assays which are easier to perform and allow for better standardization. Some groups have generated reporter cell lines that allow determination of IC 50 s by measuring HCMV spread in cell culture, reflected by luciferase activity (18) or green fluorescent protein (GFP) intensity (35). Other assays are based on recombinant viruses expressing reporter proteins, such as GFP (23), enhanced yellow fluorescent protein (12; Sarah Straschewski and Michael Winkler, personal communication), or secreted alkaline phosphatase (11).…”

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

“…We and others generated reporter cell lines for herpesviruses, including herpes simplex virus, B virus, HHV-8, and VZV (7,17,34,42,47). Two reporter cell lines for HCMV that depend on the activation of the UL54 (DNA polymerase) promoter were also published previously (13,45). However, since our transient-transfection experiments in this study and genomewide DNA microarray analyses (8) clearly demonstrated that the TRL4 promoter is severalfold stronger than the UL54 promoter, our cell line may have an advantage in terms of sensitivity.…”

A dark-to-bright reporter cell for classical swine fever virus infection

“…The basic strategy is to stably introduce genetic elements into a cell in such a way that when a particular virus enters in the cell, a virus-specific event that results in the production of an easily measurable enzyme is triggered (Olivo, 1996;Leland and Ginocchio, 2007). Cell lines expressing reporter genes inducible upon viral infection have shown to be both sensitive and specific (Olivo, 1996;Lee et al, 2004;Lutz et al, 2005;Wang et al, 2006;Ueno et al, 2006;Wu et al, 2007;Fukui et al, 2008;Li et al, 2009;Iro et al, 2009;Hossain et al, 2010;Levy et al, 2010). These reporter cells exploit the specificity of viral infection in combination with the extreme sensitivity of reporter proteins such as luciferase, chloramphenicol acetyltransferase (CAT), LacZ (b-D-galactosidase), green fluorescence protein (GFP) or secreted alkaline phosphatase (SEAP).…”