Generic placeholder image

Current HIV Research


ISSN (Print): 1570-162X
ISSN (Online): 1873-4251

Research Article

In vitro Anti-HIV-1 Activity of the Recombinant HIV-1 TAT Protein Along With Tenofovir Drug

Author(s): Maryam-Sadat Yadavar-Nikravesh, Alireza Milani , Rouhollah Vahabpour , Mehdi Khoobi , Haleh Bakhshandeh* and Azam Bolhassani*

Volume 19, Issue 2, 2021

Published on: 12 October, 2020

Page: [138 - 146] Pages: 9

DOI: 10.2174/1570162X18666201012152600

Price: $65


Background: HIV-1 TAT protein is essential for the regulation of viral genome transcription. The first exon of TAT protein has a fundamental role in the stimulation of the extrinsic and intrinsic apoptosis pathways, but its anti-HIV activity is not clear yet.

Methods: In the current study, we firstly cloned the first exon of the TAT coding sequence in the pET-24a expression vector and then protein expression was done in the Rosetta expression host. Next, the expressed TAT protein was purified by Ni-NTA column under native conditions. After that, the protein yield was determined by Bradford kit and NanoDrop spectrophotometry. Finally, the cytotoxicity effect and anti-Scr-HIV-1 activity of the recombinant TAT protein alone and along with Tenofovir drug were assessed by MTT and ELISA, respectively.

Results: The recombinant TAT protein was successfully generated in E. coli, as confirmed by 13.5% SDS-PAGE and western blotting. The protein yield was ~150-200 μg/ml. In addition, the recombinant TAT protein at a certain dose with low toxicity could suppress Scr-HIV replication in the infected HeLa cells (~30%) that was comparable with a low toxic dose of Tenofovir drug (~40%). It was interesting that the recombinant TAT protein could enhance anti-HIV potency of Tenofovir drug up to 66%.

Conclusion: Generally, a combination of TAT protein and Tenofovir drug could significantly inhibit HIV-1 replication. It will be required to determine their mechanism of action in the next studies.

Keywords: HIV-1, TAT protein, prokaryotic expression system, highly active antiretroviral therapy, tenofovir drug, anti-HIV activity.

Graphical Abstract
Garg H, Mohl J, Joshi A. HIV-1 induced bystander apoptosis. Viruses 2012; 4(11): 3020-43.
[] [PMID: 23202514]
Seitz R. German advisory committee blood (arbeitskreis blut), subgroup ‘assessment of pathogens transmissible by blood’. human immunodeficiency virus (HIV). Transfus Med Hemother 2016; 43(3): 203-22.
[] [PMID: 27403093]
Requejo HIZ. Worldwide molecular epidemiology of HIV. Rev Saude Publica 2006; 40(2): 331-45.
[] [PMID: 16583048]
Nuyen BA, Glick JL, Ferrel V, Mathews WC. HIV/AIDS. The Equal Curriculum 2020; 199-221.
Pallikkuth S, Bolivar H, Fletcher MA, et al. A therapeutic HIV-1 vaccine reduces markers of systemic immune activation and latent infection in patients under highly active antiretroviral therapy. Vaccine 2020; 38(27): 4336-45.
[] [PMID: 32387010]
Li G, De Clercq E. HIV genome-wide protein associations: a review of 30 years of research. Microbiol Mol Biol Rev 2016; 80(3): 679-731.
[] [PMID: 27357278]
Joseph AM, Ladha JS, Mojamdar M, Mitra D. Human immunodeficiency virus-1 Nef protein interacts with Tat and enhances HIV-1 gene expression. FEBS Lett 2003; 548(1-3): 37-42.
[] [PMID: 12885404]
Khairkhah N, Namvar A, Kardani K, Bolhassani A. Prediction of cross-clade HIV-1 T-cell epitopes using immunoinformatics analysis. Proteins 2018; 86(12): 1284-93.
[] [PMID: 30260061]
Hinkula J, Svanholm C, Schwartz S, et al. Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes. J Virol 1997; 71(7): 5528-39.
[] [PMID: 9188627]
Waheed AA, Freed EO. HIV type 1 Gag as a target for antiviral therapy. AIDS Res Hum Retroviruses 2012; 28(1): 54-75.
[] [PMID: 21848364]
Nagata S, Imai J, Makino G, Tomita M, Kanai A. Evolutionary analysis of HIV-1 Pol proteins reveals representative residues for viral subtype differentiation. Front Microbiol 2017; 8: 2151.
[] [PMID: 29163435]
Lu W, Chen S, Yu J, et al. The polar region of the HIV-1 envelope protein determines viral fusion and infectivity by stabilizing the gp120-gp41 association. J Virol 2019; 93(7): e02128-18.
[] [PMID: 30651369]
Melikyan GB. How entry inhibitors synergize to fight HIV. J Biol Chem 2017; 292(40): 16511-2.
[] [PMID: 28986430]
Checkley MA, Luttge BG, Freed EO. HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation. J Mol Biol 2011; 410(4): 582-608.
[] [PMID: 21762802]
Bagashev A, Sawaya BE. Roles and functions of HIV-1 Tat protein in the CNS: an overview. Virol J 2013; 10: 358.
[] [PMID: 24359561]
Feng S, Holland EC. HIV-1 tat trans-activation requires the loop sequence within tar. Nature 1988; 334(6178): 165-7.
[] [PMID: 3386755]
Jiang Y, Chai L, Fasae MB, Bai Y. The role of HIV Tat protein in HIV-related cardiovascular diseases. J Transl Med 2018; 16(1): 121.
[] [PMID: 29739413]
Frankel AD, Young JA. HIV-1: fifteen proteins and an RNA. Annu Rev Biochem 1998; 67: 1-25.
[] [PMID: 9759480]
Debaisieux S, Rayne F, Yezid H, Beaumelle B. The ins and outs of HIV-1 Tat. Traffic 2012; 13(3): 355-63.
[] [PMID: 21951552]
Stoltzfus CM. Chapter 1. Regulation of HIV-1 alternative RNA splicing and its role in virus replication. Adv Virus Res 2009; 74: 1-40.
[] [PMID: 19698894]
Balachandran A, Wong R, Stoilov P, et al. Identification of small molecule modulators of HIV-1 Tat and Rev protein accumulation. Retrovirology 2017; 14(1): 7.
[] [PMID: 28122580]
Ray N, Doms RW. HIV-1 coreceptors and their inhibitors. Curr Top Microbiol Immunol 2006; 303: 97-120.
[] [PMID: 16570858]
Buonocore L, Rose JK. Blockade of human immunodeficiency virus type 1 production in CD4+ T cells by an intracellular CD4 expressed under control of the viral long terminal repeat. Proc Natl Acad Sci USA 1993; 90(7): 2695-9.
[] [PMID: 8464877]
Dubé M, Bego MG, Paquay C, Cohen EA. Modulation of HIV-1-host interaction: role of the Vpu accessory protein. Retrovirology 2010; 7: 114.
[] [PMID: 21176220]
Marin M, Rose KM, Kozak SL, Kabat D. HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation. Nat Med 2003; 9(11): 1398-403.
[] [PMID: 14528301]
Stopak K, de Noronha C, Yonemoto W, Greene WC. HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability. Mol Cell 2003; 12(3): 591-601.
[] [PMID: 14527406]
Reddy K, Ooms M, Letko M, Garrett N, Simon V, Ndung’u T. Functional characterization of Vif proteins from HIV-1 infected patients with different APOBEC3G haplotypes. AIDS 2016; 30(11): 1723-9.
[] [PMID: 27064995]
Ogawa K, Shibata R, Kiyomasu T, et al. Mutational analysis of the human immunodeficiency virus vpr open reading frame. J Virol 1989; 63(9): 4110-4.
[] [PMID: 2474678]
González ME. The HIV-1 Vpr protein: A multifaceted target for therapeutic intervention. Int J Mol Sci 2017; 18(1): 126.
[] [PMID: 28075409]
Forouzanfar F, Ali S, Wallet C, et al. HIV-1 Vpr mediates the depletion of the cellular repressor CTIP2 to counteract viral gene silencing. Sci Rep 2019; 9(1): 13154.
[] [PMID: 31511615]
Basmaciogullari S, Pizzato M. The activity of Nef on HIV-1 infectivity. Front Microbiol 2014; 5: 232.
[] [PMID: 24904546]
Buffalo CZ, Iwamoto Y, Hurley JH, Ren X, How HIV. Nef proteins hijack membrane traffic to promote infection. J Virol 2019; 93(24): e01322-19.
[] [PMID: 31578291]
Badley AD, Pilon AA, Landay A, Lynch DH. Mechanisms of HIV-associated lymphocyte apoptosis. Blood 2000; 96(9): 2951-64.
[] [PMID: 11049971]
Tahirov TH, Babayeva ND, Varzavand K, Cooper JJ, Sedore SC, Price DH. Crystal structure of HIV-1 Tat complexed with human P-TEFb. Nature 2010; 465(7299): 747-51.
[] [PMID: 20535204]
Rice AP. The HIV-1 Tat protein: mechanism of action and target for HIV-1 cure strategies. Curr Pharm Des 2017; 23(28): 4098-102.
[] [PMID: 28677507]
Bai YL, Liu HB, Sun B, et al. HIV Tat protein inhibits hERG K+ channels: a potential mechanism of HIV infection induced LQTs. J Mol Cell Cardiol 2011; 51(5): 876-80.
[] [PMID: 21820442]
Nicoli F, Finessi V, Sicurella M, et al. The HIV-1 Tat protein induces the activation of CD8+ T cells and affects in vivo the magnitude and kinetics of antiviral responses. PLoS One 2013; 8(11): e77746.
[] [PMID: 24223723]
de Mareuil J, Carre M, Barbier P, et al. HIV-1 Tat protein enhances microtubule polymerization. Retrovirology 2005; 2: 5.
[] [PMID: 15691386]
Kim J, Kim YS. Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3. Sci Rep 2018; 8(1): 8680.
[] [PMID: 29875444]
Gibellini D, Re MC, Ponti C, et al. HIV-1 Tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-FLIP in lymphoid T cells: a potential molecular mechanism to escape TRAIL cytotoxicity. J Cell Physiol 2005; 203(3): 547-56.
[] [PMID: 15573381]
Zabihollahi R, Sadat SM, Vahabpour R, et al. Development of single-cycle replicable human immunodeficiency virus 1 mutants. Acta Virol 2011; 55(1): 15-22.
[] [PMID: 21434701]
Soleymani S, Zabihollahi R, Shahbazi S, Bolhassani A. Antiviral effects of saffron and its major ingredients. Curr Drug Deliv 2018; 15(5): 698-704.
[] [PMID: 29189153]
Romani B, Engelbrecht S, Glashoff RH. Functions of Tat: the versatile protein of human immunodeficiency virus type 1. J Gen Virol 2010; 91(Pt 1): 1-12.
[] [PMID: 19812265]
Flores SC, Marecki JC, Harper KP, Bose SK, Nelson SK, McCord JM. Tat protein of HIV-1 represses expression of manganese superoxide dismutase in HeLa cells. Proc Natl Acad Sci USA 1993; 90(16): 7632-6.
[] [PMID: 8395050]
Richard MJ, Guiraud P, Didier C, Seve M, Flores SC, Favier A. Human immunodeficiency virus type 1 Tat protein impairs selenoglutathione peroxidase expression and activity by a mechanism independent of cellular selenium uptake: consequences on cellular resistance to UV-A radiation. Arch Biochem Biophys 2001; 386(2): 213-20.
[] [PMID: 11368344]
Cota-Gomez A, Flores NC, Cruz C, et al. The human immunodeficiency virus-1 Tat protein activates human umbilical vein endothelial cell E-selectin expression via an NF-κ B-dependent mechanism. J Biol Chem 2002; 277(17): 14390-9.
[] [PMID: 11827962]
Bennasser Y, Le SY, Benkirane M, Jeang KT. Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity 2005; 22(5): 607-19.
[] [PMID: 15894278]
Shojania S, Henry GD, Chen VC, Vo TN, Perreault H, O’Neil JD. High yield expression and purification of HIV-1 Tat1-72 for structural studies. J Virol Methods 2010; 164(1-2): 35-42.
[] [PMID: 19941902]
Shi-Meng Z, Rong F, Yang T, et al. An improved strategy for efficient expression and purification of soluble HIV-1 tat protein in E. coli. Virol Sin 2009; 24: 518-28.
Kirsch T, Boehm M, Schuckert O, et al. Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 Tat protein. Protein Expr Purif 1996; 8(1): 75-84.
[] [PMID: 8812837]
Yang Y, Ma J, Song Z, Wu M. HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors11The nucleotide sequences of vectors pETAT-1/2/11/12, pNB-3/13, pHis-TAT-GFP, pHis-TAT-m-GFP and pHis-GFP have been deposited in GenBank under accession numbers AF525441–525449. FEBS Lett 2003; 532(1-2): 36-44.
[] [PMID: 12459459]
Yin J, Li G, Ren X, Herrler G. Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol 2007; 127(3): 335-47.
[] [PMID: 16959350]
Sørensen HP, Mortensen KK. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 2005; 115(2): 113-28.
[] [PMID: 15607230]
Grogan G. Practical Biotransformations: A Beginner’s Guide. 2009; pp. 1-344.
Walker JM, Raplay R. Molecular Biology and Biotechnology. 5th ed. RSC Publication 2009.
Bornhorst JA, Falke JJ. Purification of proteins using polyhistidine affinity tags. Methods Enzymol 2000; 326: 245-54.
[] [PMID: 11036646]
Mukhija S, Erni B. Purification by Ni2+ affinity chromatography, and functional reconstitution of the transporter for N-acetylglucosamine of Escherichia coli. J Biol Chem 1996; 271(25): 14819-24.
[] [PMID: 8662917]
Cihlar T, Birkus G, Greenwalt DE, Hitchcock MJ. Tenofovir exhibits low cytotoxicity in various human cell types: comparison with other nucleoside reverse transcriptase inhibitors. Antiviral Res 2002; 54(1): 37-45.
[] [PMID: 11888656]
Murphy RA, Stafford RM, Petrasovits BA, Boone MA, Valentovic MA. Establishment of HK-2 cells as a relevant model to study Tenofovir-induced cytotoxicity. Int J Mol Sci 2017; 18(3): E531.
[] [PMID: 28257038]
Cherrington JM, Allen SJW, Bischofberger N, Chen MS. Kinetic interaction of the diphosphates of 9-(2-phosphonylmethoxyethyl)adenine and other anti-HIV active purine congeners with HIV reverse transcriptase and human DNA polymerases α, β, and γ. Antivir Chem Chemother 1995; 6(4): 217-21.
Chen J, Chen R, Shen Y, et al. Efficacy and safety of lower dose tenofovir disoproxil fumarate and efavirenz versus standard dose in HIV-infected, antiretroviral-naive adults: a multicentre, randomized, noninferiority trial. Emerg Microbes Infect 2020; 9(1): 843-50.
[] [PMID: 32267205]
Cohen J. Long-acting drug acts like a short-term AIDS vaccine. Science 2020; 368(6493): 807.
[] [PMID: 32439768]
Vahabpour R, Basimi P, Roohvand F, et al. Anti-viral effects of superpositively charged mutant of green fluorescent protein. Protein Pept Lett 2019; 26(12): 930-9.
[] [PMID: 31441722]
Vahabpour R, Soleymani S, Roohvand F, Zabihollahi R, Bolhassani A. In vitro anti-viral effects of small heat shock proteins 20 and 27: A novel therapeutic approach. Curr Pharm Biotechnol 2019; 20(12): 1011-7.
[] [PMID: 31362669]
Musumeci G, Bon I, Lembo D, et al. M48U1 and Tenofovir combination synergistically inhibits HIV infection in activated PBMCs and human cervicovaginal histocultures. Sci Rep 2017; 7: 41018.
[] [PMID: 28145455]
Dabrowska A, Kim N, Aldovini A. Tat-induced FOXO3a is a key mediator of apoptosis in HIV-1-infected human CD4+ T lymphocytes. J Immunol 2008; 181(12): 8460-77.
[] [PMID: 19050264]
Bolhassani A. Target molecules and delivery vehicles for anti-HIV drugs in vitro and in vivo. Curr Pharm Des 2018; 24(29): 3393-401.
[] [PMID: 29886823]
Kumar L, Verma S, Prasad DN, Bhardwaj A, Vaidya B, Jain AK. Nanotechnology: a magic bullet for HIV AIDS treatment. Artif Cells Nanomed Biotechnol 2015; 43(2): 71-86.
[] [PMID: 24564348]
Jiang XC, Gao JQ. Exosomes as novel bio-carriers for gene and drug delivery. Int J Pharm 2017; 521(1-2): 167-75.
[] [PMID: 28216464]
Sarkar S, Dasgupta AK. Microparticle of drug and nanoparticle: a biosynthetic route. Pharmacol Res Perspect 2015; 3(5): e00188.
[] [PMID: 26516592]
Kotmakçı M, Bozok Çetintaş V. Extracellular vesicles as natural nanosized delivery systems for small-molecule drugs and genetic material: Steps towards the future nanomedicines. J Pharm Pharm Sci 2015; 18(3): 396-413.
[] [PMID: 26517135]
van der Meel R, Fens MH, Vader P, van Solinge WW, Eniola-Adefeso O, Schiffelers RM. Extracellular vesicles as drug delivery systems: lessons from the liposome field. J Control Release 2014; 195: 72-85.
[] [PMID: 25094032]
van Dommelen SM, Vader P, Lakhal S, et al. Microvesicles and exosomes: opportunities for cell-derived membrane vesicles in drug delivery. J Control Release 2012; 161(2): 635-44.
[] [PMID: 22138068]
Vader P, Mol EA, Pasterkamp G, Schiffelers RM. Extracellular vesicles for drug delivery. Adv Drug Deliv Rev 2016; 106(Pt A): 148-56.
[] [PMID: 26928656]
Soleymani S, Yari F, Bolhassani A, Bakhshandeh H. Platelet microparticles: An effective delivery system for anti-viral drugs. J Drug Deliv Sci Technol 2019; 51: 290-6.

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy