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Abstract

Human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter-mediated gene expression is regulated by the viral Tat protein that relieves a block to viral transcription elongation after binding with a viral hairpin loop RNA structure called the trans-activation-responsive region (TAR). Tat protein significantly up-regulates viral genome transcription and hence it has long been considered a potential target for antiretrovirals. Here we report the construction of a plasmid containing an HIV-1 LTR-driven reporter cassette with a colinear tat gene under control of a viral promoter and thus conditionally configured for constitutive expression of reporter genes. Inhibition of luciferase reporter expression in a cell line harboring the plasmid in the presence of tat-targeted shRNA confirmed the specificity of the assay and a dose-dependent reporter activity inhibition by the fluoroquinoline derivative K-37, a class of small RNA binding molecule that inhibits Tat and other RNA-dependent transactivations, further validated the method. Subsequently we also made a lentiviral vector (LV) containing the same transcription units and derived a stable cell line using the said LV and similar dose-dependent inhibition was documented using K-37. This quick and sensitive reporter-based method is the simplest screening assay for putative inhibitors of HIV-1 Tat-induced LTR-driven gene expression requiring test material addition as the only manipulation.

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References

Berkhout

, Silverman

, Jeang

. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell, 1989; 59:273–282.

Feinberg

, Baltimore

, Frankel

. The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation. Proc Natl Acad Sci USA, 1991; 88:4045–4049.

Harrich

, Ulich

, Garcia-Martinez

et al. Tat is required for efficient HIV-1 reverse transcription. EMBO J, 1997; 16:1224–1235.

Myers

, Korber

, Foley

. Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory: Los Alamos, NM, 1998; III-11–III-26.

Dingwall

, Ernberg

, Gait

et al. Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. Proc Natl Acad Sci USA, 1989; 86:6925–6929.

Mavankal

, Ignatius Ou

, Oliver

et al. Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II. Proc Natl Acad Sci USA, 1996; 93:2089–2094.

Davidson

, Leeper

, Athanassiou

et al. Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein. Proc Natl Acad Sci USA, 2009; 106:11931–11936.

, Liu

, Hsu

et al. HIV-1 Tat and host AFF4 recruit two transcription elongation factors into a bifunctional complex for coordinated activation of HIV-1 transcription. Mol Cell, 2010; 38:428–438.

Sobhian

, Laguette

, Yatim

et al. HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP. Mol Cell, 2010; 38:439–451.

10.

, Zhou

. New insights into the control of HIV-1 transcription: When tat meets the 7SK snRNP and super elongation complex (SEC) J Neuroimmune Pharmacol, 2011; 6:260–268.

11.

Hsu

, Schutt

, Holly

et al. Inhibition of HIV replication in acute and chronic infections in vitro by a Tat antagonist. Science, 1991; 254:1799–1802.

12.

Gatignol

, Jeang

. Tat as a transcriptional activator and a potential therapeutic target for HIV-1. Adv Pharmacol, 2000; 48:209–227.

13.

Baba

. Recent status of HIV-1 gene expression inhibitors. Antiviral Res, 2006; 71:301–306.

14.

Richter

, Parolin

, Gatto

et al. Inhibition of human immunodeficiency virus type 1 tat-trans-activation-responsive region interaction by an antiviral quinolone derivative. Antimicrob Agents Chemother, 2004; 48:1895–1899.

15.

Koseki

, Ohkawa

, Yamamoto

et al. A simple assay system for examination of the inhibitory potential in vivo of decoy RNAs, ribozymes and other drugs by measuring the Tat-mediated transcription of a fusion gene composed of the long terminal repeat of HIV-1 and a gene for luciferase. J Control Release, 1998; 53:159–173.

16.

Jeeninga

, Hoogenkamp

, Armand-Ugon

et al. Functional differences between the long terminal repeat transcriptional promoters of human immunodeficiency virus type 1 subtypes A through G. J Virol, 2000; 74:3740–3751.

17.

Corish

, Tyler-Smith

. Attenuation of green fluorescent protein half-life in mammalian cells. Prot Engineer, 1999; 12:1035–1040.

18.

Ravi

, Mitra

. HIV-1 long terminal repeat promoter regulated dual reporter: Potential use in screening of transcription modulators. Anal Biochem, 2007; 360:315–317.

19.

Ranga

, Shankarappa

, Siddappa

et al. Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine. J Virol, 2004; 78:2586–2590.

20.

Pei

, Tuschl

. On the art of identifying effective and specific siRNAs. Nature Methods, 2006; 3:670–676.

21.

Hauber

, Perkins

, Heimer

et al. Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc Natl Acad Sci USA, 1987; 84:6364–6368.

22.

Santhosh

, Tamhane

, Kamat

et al. A lentiviral vector with novel multiple cloning sites: Stable transgene expression in vitro and in vivo. Biochem Biophys Res Commun, 2008; 371:546–550.

23.

Baba

, Okamoto

, Kawamura

et al. Inhibition of human immunodeficiency virus type 1 replication and cytokine production by fluoroquinoline derivatives. Mol Pharmacol, 1998; 53:1097–1103.

24.

Okamoto

, Cujec

, Okamoto

et al. Inhibition of the RNA-dependent transactivation and replication of human immunodeficiency virus type 1 by a fluoroquinoline derivative K-37. Virology, 2000; 272:402–408.

25.

Alam

, Cook

. Reporter genes: Application to the study of mammalian gene transcription. Anal Biochem, 1990; 188:245–254.

26.

Daelemans

, De Clercq

, Vandamme

. A quantitative GFP-based bioassay for the detection of HIV-1 Tat transactivation inhibitors. J Virol Methods, 2001; 96:183–188.

27.

Emmanuel

, Christine

, James

et al. Nuclear factor 90 (NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1. Retrovirology, 2007; 4:41–44.

28.

Jiang

, Lin

, Chen

. Inhibition of HIV-1 Tat-mediated transactivation by quinacrine and chloroquine. Biochem Biophys Res Commun, 1996; 226:1–7.

29.

Lisziewicz

, Sun

, Smythe

et al. Inhibition of human immunodeficiency virus type 1 replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS. Proc Natl Acad Sci USA, 1993; 90:8000–8004.

30.

Yang

. Discoveries of Tat-TAR interaction inhibitors for HIV-1. Curr Drug Targets Infect Disorders, 2005; 5:433–444.

31.

Kira

, Merin

, Baba

et al. Anti-Tat MTT assay: A novel anti-HIV drug screening system using the viral regulatory network of replication. AIDS Res Hum Retroviruses, 1995; 11:1359–1366.

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Short Communication: A Single Step Assay for Rapid Evaluation of Inhibitors Targeting HIV Type 1 Tat-Mediated Long Terminal Repeat Transactivation

Abstract

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