Activity of the dietary antioxidant ergothioneine in a virus gene-based assay for inhibitors of HIV transcription.
The "Long Terminal Repeat" (LTR) of HIV-1 is the target of cellular transcription factors such as NF-kappaB, and serves as the promoter-enhancer for the viral genome when integrated in host DNA. Various LTR-reporter gene constructs have been used for in vitro studies of activators or inhibitors of HIV-1 transcription, e.g., to show that antioxidants such as lipoic acid and selenium inhibit NF-kappaB-dependent HIV-1 LTR activation. One such construct is the pHIVlacZ plasmid, with the HIV-1 LTR driving expression of the lacZ gene (encoding beta-galactosidase, beta-gal). Typically, for inhibitor screening, cells transfected with pHIVlacZ are activated using tumor necrosis factor-alpha (TNF-alpha), and the colorimetric o-nitrophenol assay is used to assess changes in beta-gal activity. A variant of this assay was developed as described here, in which LTR activation was induced by pro-fs, a novel HIV-1 gene product encoded via a -1 frameshift from the protease gene. Cotransfection of cells with pHIVlacZ along with a pro-fs construct produced a significant increase in beta-gal activity over controls. L-ergothioneine dose dependently inhibited both TNF-alpha-mediated and pro-fs-mediated increases in beta-gal activity, with an IC50 of about 6 mM. Thus antioxidant strategy involving ergothioneine derived from food plants might be of benefit in chronic immunodeficiency diseases.
Xiao
Lianchun
L
Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA.
Zhao
Lijun
L
Li
Ting
T
Hartle
Diane K
DK
Aruoma
Okezie I
OI
Taylor
Ethan Will
EW
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002460:Cell Line; D019587:Dietary Supplements; D004305:Dose-Response Relationship, Drug; D004880:Ergothioneine / Q000494:pharmacology*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006678:HIV / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000975:Antioxidants; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D004880:Ergothioneine