Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0.
The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-h period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0.
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, United States.
Angela M
AM
Fowler
Microscopy and Analytical Imaging Laboratory, University of Kansas, Lawrence, KS 66045, United States.
Heather E
HE
Shinogle
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, United States. Electronic address: ddavido@ku.edu.
David J
DJ
Davido
D003584:Cytological Techniques / Q000379:methods*; D005470:Fluorometry; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D015533:Transcriptional Activation; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D017874:Immediate-Early Proteins; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1
Cell-based assay; GFP; HSV; ICP0; Viral transcription