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, Patrizia Caposio, M. Mondini, S. Landolfo, G. Gribaudo

Research output: Contribution to journalArticle

14 Citations (Scopus)

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-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.

Original languageEnglish (US)
Pages (from-to)1791-1801
Number of pages11
JournalJournal of Applied Microbiology
Volume105
Issue number6
DOIs
StatePublished - Dec 2008
Externally publishedYes

Fingerprint

Immediate-Early Proteins
Cytomegalovirus Infections
Viral Proteins
Cytomegalovirus
Fluorescence
Proteins
Antiviral Agents
Oligodeoxyribonucleotides
Reporter Genes
Genes
Transcription Factors
Clone Cells
Cell Line

Keywords

  • Antivirals
  • Cell-based assay
  • Early gene promoters
  • Enhanced green fluorescence protein
  • Human cytomegalovirus
  • Immediate-early 2 activity
  • Indicator cells

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Biotechnology

Cite this

New cell-based indicator assays for the detection of human cytomegalovirus infection and screening of inhibitors of viral immediate-early 2 protein activity. / Luganini, A.; Caposio, Patrizia; Mondini, M.; Landolfo, S.; Gribaudo, G.

In: Journal of Applied Microbiology, Vol. 105, No. 6, 12.2008, p. 1791-1801.

Research output: Contribution to journalArticle

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