Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine–adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future.
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References
1.
AgostoL.M., YuJ.J., DaiJ.et al.2007. HIV-1 integrates into resting CD4+ T cells even at low inoculums as demonstrated with an improved assay for HIV-1 integration. Virology, 368:60–72.
2.
BalakrishnanM., JonssonC.B.1997. Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions. J. Virol., 71:1025–1035.
3.
BarbosaP., CharneauP., DumeyN.et al.1994. Kinetic analysis of HIV-1 early replicative steps in a coculture system. AIDS Res. Hum. Retroviruses, 10:53–59.
4.
BohjanenP.R., ColvinR.A., PuttarajuM.et al.1996. A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription. Nucleic Acids Res., 24:3733–3738.
5.
BugreevD.V., BaranovaS., ZakharovaO.D.et al.2003. Dynamic, thermodynamic, and kinetic basis for recognition and transformation of DNA by human immunodeficiency virus type 1 integrase. Biochemistry, 42:9235–9247.
6.
BushmanF.D., CraigieR.1991. Activities of human immunodeficiency virus (HIV) integration protein in vitro: Specific cleavage and integration of HIV DNA. Proc. Natl. Acad. Sci. U.S.A., 88:1339–1343.
7.
CavertW., NotermansD.W., StaskusK.et al.1997. Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection. Science, 276:960–964.
8.
CharpentierC., KarmochkineM., LaureillardD.et al.2008. Drug resistance profiles for the HIV integrase gene in patients failing raltegravir salvage therapy. HIV Med., 9:765–770.
9.
ChenL.F., HoyJ., LewinS.R.2007. Ten years of highly active antiretroviral therapy for HIV infection. Med. J. Aust., 186:146–151.
10.
CohliH., FanB., JoshiR.L.et al.1994. Inhibition of HIV-1 multiplication in a human CD4+ lymphocytic cell line expressing antisense and sense RNA molecules containing HIV-1 packaging signal and Rev response element(s)Antisense Res. Dev., 4:19–26.
11.
De ClercqE.2001. New developments in anti-HIV chemotherapy. Curr. Med. Chem., 8:1543–1572.
12.
DelelisO., MaletI., NaL.et al.2009. The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation. Nucleic Acids Res., 37:1193–1201.
13.
DeprezE., BarbeS., KolaskiM.et al.2004. Mechanism of HIV-1 integrase inhibition by styrylquinoline derivatives in vitro. Mol. Pharmacol., 65:85–98.
14.
DonehowerL.A., VarmusH.E.1984. A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration. Proc. Natl. Acad. Sci. U.S.A., 81:6461–6465.
15.
DreierB., BeerliR.R., SegalD.J.et al.2001. Development of zinc finger domains for recognition of the 5′-ANN-3′ family of DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem., 276:29466–29478.
16.
DullT., ZuffereyR., KellyM.et al.1998. A third-generation lentivirus vector with a conditional packaging system. J. Virol., 72:8463–8471.
17.
EveringT.H., MarkowitzM.2008. Raltegravir: An integrase inhibitor for HIV-1. Expert Opin. Investig. Drugs, 17:413–422.
18.
FlexnerC.2007. HIV drug development: The next 25 years. Nat. Rev. Drug Discov., 6:959–966.
19.
GrantP., ZolopaA.2008. Integrase inhibitors: A clinical review of raltegravir and elvitegravir. J. HIV Ther., 13:36–39.
20.
GuptaR.K., PillayD.2007. HIV resistance and the developing world. Int. J. Antimicrob. Agents, 29:510–517.
21.
HanY., LassenK., MonieD.et al.2004. Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes. J. Virol., 78:6122–6133.
22.
HicksC., GulickR.M., RoquebertB.et al.2009. Raltegravir: The first HIV type 1 integrase inhibitor: Selection of the Q148R integrase inhibitor resistance mutation in a failing raltegravir containing regimen. Clin. Infect. Dis., 48:931–939.
23.
JurriaansS., de RondeA., DekkerJ.et al.1992. Analysis of human immunodeficiency virus type 1 LTR–LTR junctions in peripheral blood mononuclear cells of infected individuals. J. Gen. Virol., 73:1537–1541.
24.
KatzR.A., SkalkaA.M.1994. The retroviral enzymes. Annu. Rev. Biochem., 63:133–173.
25.
KatzmanM., SudolM.1996. Influence of subterminal viral DNA nucleotides on differential susceptibility to cleavage by human immunodeficiency virus type 1 and visna virus integrases. J. Virol., 70:9069–9073.
26.
KimS.Y., ByrnR., GroopmanJ.et al.1989. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression. J. Virol., 63:3708–3713.
27.
LaFeminaR.L., CallahanP.L., CordingleyM.G.1991. Substrate specificity of recombinant human immunodeficiency virus integrase protein. J. Virol., 65:5624–5630.
28.
LeeN.S., DohjimaT., BauerG.et al.2002. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol., 20:500–505.
29.
LevyJ.A.2007. Step involved in HIV:cell interaction and virus entry. HIV and the Pathogenesis of AIDS. ASM Press: Washington, D.C., 77.
30.
Levy-MintzP., DuanL., ZhangH.et al.1996. Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase. J. Virol., 70:8821–8832.
LiemS.E., RamezaniA., LiX.et al.1993. The development and testing of retroviral vectors expressing trans-dominant mutants of HIV-1 proteins to confer anti-HIV-1 resistance. Hum. Gene Ther., 4:625–634.
LuX., YuQ., BinderG.K.et al.2004. Antisense-mediated inhibition of human immunodeficiency virus (HIV) replication by use of an HIV type 1-based vector results in severely attenuated mutants incapable of developing resistance. J. Virol., 78:7079–7088.
35.
MarascoW.A., HaseltineW.A., ChenS.Y.1993. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc. Natl. Acad. Sci. U.S.A., 90:7889–7893.
36.
MarchettiA., ButtittaF., MiyazakiS.et al.1995. Int-6, a highly conserved, widely expressed gene, is mutated by mouse mammary tumor virus in mammary preneoplasia. J. Virol., 69:1932–1938.
37.
MbisaJ.L., MartinS.A., CaneP.A.2011. Patterns of resistance development with integrase inhibitors in HIV. Infect. Drug Resist., 4:65–76.
38.
O'DohertyU., SwiggardW.J., JeyakumarD.et al.2002. A sensitive, quantitative assay for human immunodeficiency virus type 1 integration. J. Virol., 76:10942–10950.
39.
PalellaF.J.Jr., DelaneyK.M., MoormanA.C.et al.HIV Outpatient Study Investigators. 1998. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N. Engl. J. Med., 338:853–860.
40.
PauzaC.D.1990. Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination. Virology, 179:886–889.
41.
PauzaC. D., TrivediP., McKechnieT. S.et al.1994. 2-LTR circular viral DNA as a marker for human immunodeficiency virus type 1 infection in vivo. Virology, 205:470–478.
42.
RamezaniA., MaX.Z., NazariR.et al.2002. Development and testing of retroviral vectors expressing multimeric hammerhead ribozymes targeted against all major clades of HIV-1. Front. Biosci., 7:a29–a36.
43.
ReismanD., SugdenB.1986. Trans-activation of an Epstein–Barr viral transcriptional enhancer by the Epstein–Barr viral nuclear antigen 1. Mol. Cell. Biol., 6:3838–3846.
44.
ReynoldsL., UllmanC., MooreM.et al.2003. Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proc. Natl. Acad. Sci. U.S.A., 100:1615–1620.
45.
SakkhachornphopS., JiranusornkulS., KodchakornK.et al.2009. Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions. Protein Sci., 18:2219–2230.
46.
SchragerL.K., D'SouzaM.P.1998. Cellular and anatomical reservoirs of HIV-1 in patients receiving potent antiretroviral combination therapy. JAMA, 280:67–71.
47.
SegalD.J., GoncalvesJ., EberhardyS.et al.2004. Attenuation of HIV-1 replication in primary human cells with a designed zinc finger transcription factor. J. Biol. Chem., 279:14509–14519.
48.
ShaheenF., DuanL., ZhuM.et al.1996. Targeting human immunodeficiency virus type 1 reverse transcriptase by intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle. J. Virol., 70:3392–3400.
49.
ShermanP.A., FyfeJ.A.1990. Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity. Proc. Natl. Acad. Sci. U.S.A., 87:5119–5123.
50.
StaunstrupN. H., MoldtB., MatesL.et al.2009. Hybrid lentivirus–transposon vectors with a random integration profile in human cells. Mol. Ther., 17:1205–1214.
51.
SuK., WangD., YeJ.et al.2009. Site-specific integration of retroviral DNA in human cells using fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc-finger protein E2C. Methods, 47:269–276.
52.
TamhaneM., AkkinaR.2008. Stable gene transfer of CCR5 and CXCR4 siRNAs by Sleeping Beauty transposon system to confer HIV-1 resistance. AIDS Res. Ther., 5:16.
53.
TanW., ZhuK., SegalD.J.et al.2004. Fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc finger protein E2C direct integration of viral DNA into specific sites. J. Virol., 78:1301–1313.
54.
TesioM., GammaitoniL., GunettiM.et al.2008. Sustained long-term engraftment and transgene expression of peripheral blood CD34+ cells transduced with third-generation lentiviral vectors. Stem Cells, 26:1620–1627.
55.
TewariD., NotkinsA.L., ZhouP.2003. Inhibition of HIV-1 replication in primary human T cells transduced with an intracellular anti-HIV-1 p17 antibody gene. J. Gene Med., 5:182–189.
56.
van den EntF.M., VinkC., PlasterkR.H.1994. DNA substrate requirements for different activities of the human immunodeficiency virus type 1 integrase protein. J. Virol., 68:7825–7832.
57.
VercruysseT., PardonE., VanstreelsE.et al.2010. An intrabody based on a llama single-domain antibody targeting the N-terminal α-helical multimerization domain of HIV-1 Rev prevents viral production. J. Biol. Chem., 285:21768–21780.
58.
VinkC., PlasterkR.H.1993. The human immunodeficiency virus integrase protein. Trends Genet., 9:433–438.
59.
VinkC., van GentD. C., ElgersmaY.et al.1991. Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage. J. Virol., 65:4636–4644.
60.
WhitcombJ.M., KumarR., HughesS.H.1990. Sequence of the circle junction of human immunodeficiency virus type 1: Implications for reverse transcription and integration. J. Virol., 64:4903–4906.
61.
YatesJ.L., WarrenN., SugdenB.1985. Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells. Nature, 313:812–815.
62.
YoshinagaT., FujiwaraT.1995. Different roles of bases within the integration signal sequence of human immunodeficiency virus type 1 in vitro. J. Virol., 69:3233–3236.
63.
ZuffereyR., DullT., MandelR.J.et al.1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol., 72:9873–9880.
