Abstract
Human immunodeficiency virus regulatory protein Rev (regulator of viral expression) is translated from a monocistronic transcript produced early in the viral replication cycle. Rev binds to the cis-acting, highly structured viral RNA sequence Rev response element (RRE) and the Rev–RRE complex primarily controls nucleocytoplasmic transport of viral RNAs. Inhibition of Rev–RRE interaction therefore is an attractive target to block viral transport. We have developed a stable cell line carrying a lentiviral vector harboring a rev gene and a co-linear Rev-dependent GFP/luciferase reporter gene cassette and thus constitutively expressing the reporter proteins. Dose-dependent luciferase activity inhibition in the indicator cell line by known small molecule inhibitors Proflavin and K37 established the specificity of the assay. This novel single step assay, that involves use of very small amount of reagents/cells and addition of test material as the only manipulation, can therefore be useful for screening therapeutically potential Rev–RRE interaction inhibitors.
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Acknowledgments
We thank Dr. Debashis Mitra, National Cell Science Centre, Pune, for providing pcDNA-Rev and pINDIE-C1, Dr. Didier Trono, Swiss Institute of Technology, Lausanne, Switzerland for the pMD.G, and Dr. Pierre Charneau, Pasteur Institute, Paris, France for pTEG. The HIV-1 Rev antibody (Cat #7376) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 Rev Monoclonal Antibody (1G7) from Dr. Anne Marie Szilvay. The study was supported by institutional core grant. SR and AGC were supported by research fellowships from ACTREC, Tata Memorial Centre.
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Supplementary Fig. 1
Fluorescent microscopy imaging (Upper panel) and corresponding Flow cytometry profile (Lower panel) of EGFP expression in (a) HEK293 cells, (b) control cell line and (c) indicator cell line (TIF 597 kb)
Supplementary Fig. 2
Rev-dependent reporter (luciferase) expression augmentation in the indicator cell line compared to the control cell line. Columns and error bars are mean + sd (n = 3) (TIF 76 kb)
Supplementary Fig. 3
A graphical abstract of the assay principle (TIF 245 kb)
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Raina, S., Chande, A.G., Baba, M. et al. A reporter based single step assay for evaluation of inhibitors targeting HIV-1 Rev–RRE interaction. VirusDis. 25, 101–106 (2014). https://doi.org/10.1007/s13337-013-0166-8
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DOI: https://doi.org/10.1007/s13337-013-0166-8