ORIGINAL ARTICLE New cell-based indicator assays for the detection of human cytomegalovirus infection and screening of inhibitors of viral immediate-early 2 protein activity A. Luganini 1 , P. Caposio 1 , M. Mondini 2 , S. Landolfo 1 and G. Gribaudo 1 1 Department of Public Health and Microbiology, University of Torino, Torino, Italy 2 Department of Clinical and Experimental Medicine, University of Piemonte Orientale, Novara, Italy Introduction Human cytomegalovirus (HCMV) is a ubiquitous mem- ber of the b-herpesvirus family and a significant human opportunist pathogen that usually causes asymptomatic infections in healthy individuals. However, primary infec- tion or reactivation of latent virus in immunocompro- mised individuals can lead to severe life-threatening disease (Landolfo et al. 2003; Sinclair and Sissons 2006). Typical of herpesviruses, HCMV gene expression is tem- porally regulated after infection and can be divided into immediate-early (IE), early (E) and late (L) phases. Viral IE genes encode the transcriptional regulators IE1 p72 (IE1) and IE2 p86 (IE2), which are required for the subsequent expression of E and L genes (Mocarski and Courcelle 2001; Landolfo et al. 2003; Meier and Stinski 2006). Regulation of IE gene expression is the key to pro- ductive replication and latency because of their ability to transactivate and ⁄ or repress both viral and cellular genes (Sinclair and Sissons 2006). Manipulation of HCMV gen- ome by BAC-technology mutagenesis has confirmed the indispensable role of IE2 in viral replication and its cru- cial functions in transactivation of viral E and L genes and in auto-repression (Meier and Stinski 2006). More- over, there is growing evidence that IE2 alone, or in syne- rgism with IE1, plays a direct role in the pathogenesis of HCMV infection, by inducing a broad dysregulation of host gene expression. This leads to changes in host cell Keywords antivirals, cell-based assay, early gene promoters, enhanced green fluorescence protein, human cytomegalovirus, immediate- early 2 activity, indicator cells. Correspondence Giorgio Gribaudo, Department of Public Health and Microbiology, University of Torino, Via Santena 9, 10126 Turin, Italy. E-mail: giorgio.gribaudo@unito.it 2008 ⁄ 0147: received 25 January 2008, revised 9 April 2008 and accepted 15 April 2008 doi:10.1111/j.1365-2672.2008.03927.x Abstract 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-depen- dent 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. Journal of Applied Microbiology ISSN 1364-5072 ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1791–1801 1791