AIMS: Expression of early (E) genes of human cytomegalovirus (HCMV) is stimulated cooperatively by the activities of host cell transcription factors and the viral immediate-early 2 (IE2) protein. Taking advantage of the IE2-dependent inducibility of E gene promoters, in this study, we generated cell-based assays in which the expression of the enhanced green fluorescence protein (EGFP) reporter gene was driven by the UL54 or UL112/113 E promoters. METHODS AND RESULTS: Cell clones derived from a stably transfected human cell line permissive to HCMV replication showed a specific and inducible dose- and time-dependent EGFP response to HCMV infection. The sensitivity of these indicator cells for detecting infectious particles of clinical isolates of HCMV was comparable to that of a conventional plaque assay. The HCMV-induced EGFP expression was completely prevented by treatment of indicator cells with fomivirsen, an antisense oligodeoxynucleotide designed to block IE2 expression, and this inhibitory activity was also observed when the IE2 protein alone was constitutively expressed in EGFP indicator cells. CONCLUSIONS: The EGFP-based cell assays have proved to be a rapid, sensitive, quantitative and specific system for detection of HCMV and selection of antivirals. SIGNIFICANCE AND IMPACT OF THE STUDY: These new cell-based assays can be exploited as functional assays to detect infectious HCMV particles, as well as to screen antiviral compounds that interfere with IE2 activity.
AIMS: Expression of early (E) genes of human cytomegalovirus (HCMV) is stimulated cooperatively by the activities of host cell transcription factors and the viral immediate-early 2 (IE2) protein. Taking advantage of the IE2-dependent inducibility of E gene promoters, in this study, we generated cell-based assays in which the expression of the enhanced green fluorescence protein (EGFP) reporter gene was driven by the UL54 or UL112/113 E promoters. METHODS AND RESULTS: Cell clones derived from a stably transfected human cell line permissive to HCMV replication showed a specific and inducible dose- and time-dependent EGFP response to HCMV infection. The sensitivity of these indicator cells for detecting infectious particles of clinical isolates of HCMV was comparable to that of a conventional plaque assay. The HCMV-induced EGFP expression was completely prevented by treatment of indicator cells with fomivirsen, an antisense oligodeoxynucleotide designed to block IE2 expression, and this inhibitory activity was also observed when the IE2 protein alone was constitutively expressed in EGFP indicator cells. CONCLUSIONS: The EGFP-based cell assays have proved to be a rapid, sensitive, quantitative and specific system for detection of HCMV and selection of antivirals. SIGNIFICANCE AND IMPACT OF THE STUDY: These new cell-based assays can be exploited as functional assays to detect infectious HCMV particles, as well as to screen antiviral compounds that interfere with IE2 activity.
Authors: Anna Katharina Maier; Raimund Jung; Clarissa Villinger; Axel Schubert; Paul Walther; Christian Sinzger; Diana Lieber Journal: PLoS One Date: 2017-01-06 Impact factor: 3.240