Generation and use of recombinant reporter viruses for study of herpes simplex virus infections in vivo.
It has become increasingly clear that the fate of herpes simplex virus (HSV) infections involves complex interactions between the virus and the specific cell types that comprise the tissues of the animal host. No reliable cell culture system for studying the establishment of latency and reactivation exists, and therefore these studies must be performed within animal models. One difficulty in elucidating the molecular regulation of these events is in determining the transcriptional activity of key viral genes during different stages of the infection in vivo. The heterogeneous cell types comprising infected tissues make PCR analysis of tissue homogenates difficult to interpret. The need to characterize expression of multiple transcriptional markers reliably and quantitatively at the level of individual cells is therefore key to determining the interplay between viral and cellular genes during latency and reactivation. Here we discuss the construction and evaluation of HSV reporter viruses that have been used in these analyses. HSV-1 recombinants have been engineered with representative viral promoters driving beta-galactosidase as a reporter. Methodology used to evaluate the levels of gene expression using (1) quantitative enzyme assays, (2) precipitatable substrate assays to localize the positive cells, and (3) immunohistochemistry and fluorescence assays to look at colocalization of markers during in vivo infection is presented. In addition to studying the molecular pathogenesis of HSV, the application of similar reporter viruses to evaluate promoters for targeting various differentiated tissues will be useful in developing these viruses as potential vectors for gene therapy.
Bloom
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Department of Microbiology, Arizona State University, Tempe, Arizona, 85287-2701, USA. dcbloom@asu.edu
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D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D001921:Brain / Q000166:cytology / Q000821:virology; D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics; D005727:Ganglia, Spinal / Q000166:cytology / Q000821:virology; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000472:pathogenicity*; D007150:Immunohistochemistry; D051379:Mice; D008815:Mice, Inbred Strains; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D011995:Recombination, Genetic / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000956:Antigens, Viral; D004279:DNA, Viral; D001616:beta-Galactosidase