Antiviral strategies to inhibit HIV-1 replication have included the generation of gene products that provide the intracellular inhibition of an essential viral protein or RNA. When used in conjunction with the HIV-1 long terminal repeat (LTR), an inducible promoter dependent on the virus-encoded trans-activator (tat), relatively high background activity is still observed in the absence of tat (Caruso & Klatzmann, 1992; Dinges et al., 1995). In order to circumvent this problem, we used the Cre/loxP (ON/OFF) recombination system as a tool for our investigation. In the present study, we constructed a loxP-cassette vector with the ribozyme (Rz) expression portion under the control of the tRNAiMet promoter between two loxP sequences (plox-Rz-U5). We also constructed an HIV-1 LTR promoter-driven Cre recombinase gene (pLTR-Cre). These vectors were triple-transfected into HeLa CD4 cells with the HIV-1 pseudo-type viral expression vector. Basal activity was not detectable before HIV-1 infection. The LTR-dependent Cre protein product in HIV-1 infected HeLa CD4 cells expressed the ribozyme by inducing loxP homologous recombination, which strongly inhibited the HIV-1 gene expression. These results demonstrate the potential of an anti-ribozyme with the Cre/loxP system for controlling HIV-1 infection via gene therapy.
AbremskiKHoessR & StenbergN (1983). Studies on the properties of P1 site-specific recombination: Evidence for topologically unlinked products following recombination. Cell32: 1301–1311.
2.
AbremskiK & HoessR (1984). Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein. Journal of Biological Chemistry259: 1509–1514.
3.
AdachiAGendelmanHEKoenigSFolksTWilleyRRabsonA & MartinMA (1986). Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. Journal of Virology59: 284–291.
4.
AdamsDEBliskaJB & CozzarelliNR (1992). Cre-lox recombination in Escherichia coli cells. Mechanistic differences from the in vitro reaction. Journal of Molecular Biology226: 661–673.
5.
AntonM & GrahamFL (1995). Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: A molecular switch for control of gene expression. Journal of Virology69: 4600–4606.
CarusoM & KlatzmannD (1992). Selective killing of CD4 cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population. Proceedings of the National Academy of Sciences USA89: 182–186.
8.
DahmSC & UhlenbeckOC (1991). Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry30: 9464–9469.
9.
DahmSCDerrickWB & UhlenbeckOC (1993). Evidence for the role of solvated metal hydroxide in the hammerhead cleavage mechanism. Biochemistry32: 13040–13045.
10.
DingesMMCookDRKingJCurielTJZhangXQ & HarrisonGS (1995). HIV-regulated diphtheria toxin A chain gene confers long-term protection against HIV type 1 infection in the human promonocytic cell line U937. Human Gene Therapy6: 1437–1445.
11.
DuZRicordCInverardiLPodackE & PastoriRL (1998). Efficient ex vivo inhibition of perforin and Fas ligand expression by chimeric tRNA-hammerhead ribozymes. Human Gene Therapy9: 1551–1560.
12.
DropulicB & JeangK (1994). Gene therapy for human immunodeficiency virus infection: Genetic antiviral strategies and targets for intervention. Human Gene Therapy5: 927–939.
13.
el KharroubiA & VerdinE (1994). Protein-DNA interactions within DNase I-hypersensitive sites located downstream of the HIV-1 promoter. Journal of Biological Chemistry269: 19916–19924.
14.
FlowersCCWoffendinCPetryniakJYangS & NabelGJ (1997). Inhibition of recombinant human immunodeficiency virus type 1 replication by a site-specific recombinase. Journal of Virology71: 2685–2692.
15.
FukushigeS & SauerB (1992). Genomic Targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proceedings of the National Academy of Sciences USA89: 7905–7909.
16.
GaynorR (1992). Cellular transcription factors involved in the regulation of HIV-1 gene expression. AIDS6: 6347–6363.
17.
GuHZouYR & RajewskyK (1993). Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell73: 1155–1164.
18.
GuHMarthJDOrbanPCMossmannH & RajewskyK (1994). Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science265: 103–106.
19.
Guerrier-TakadaCGardinerKMarshTPaceN & AltmanS (1983). The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell35: 849–857.
20.
HoessRHZieseM & SternbergN (1982). P1 site-specific recombination: Nucleotide sequence of the recombining sites. Proceedings of the National Academy of Sciences USA79: 3398–3402.
21.
HoessRH & AbremskiK (1984). Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP. Proceedings of the National Academy of Sciences USA81: 1026–1029.
22.
HoessR & AbremskiK (1985). Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. Journal of Molecular Biology181: 351–362.
23.
KeithGHeitzlerJEl AdlouniCGlasserALFixCDesgresJ & DirheimerG (1993). The primary structure of cytoplasmic initiator tRNA(Met) from Schizosaccharomyces pombe. Nucleic Acids Research21: 2949.
24.
KrugerKGrabowskiPJZaugAJSandsJGottschlingDE & CechTR (1982). Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell31: 147–157.
LaksoMSauerBMosingerBJrLeeEJManningRWYuSHMulderKL & WestphalH (1992). Targeted oncogene activation by site-specific recombination in transgenic mice. Proceedings of the National Academy of Sciences USA89: 6232–6236.
27.
LarssonSHotchkissGSuJKebedeTAndangMNyholmTJohanssonBSonnerborgAVahlneABrittonS & Ahrlund-RichterL (1996). A novel ribozyme target site located in the HIV-1 nef open reading frame. Virology219: 161–169.
28.
LottWBPontiusBW & von HippelPH (1998). A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate. Proceedings of the National Academy of Sciences USA95: 542–547.
29.
OrbanPCChuiD & MarthJD (1992). Tissue- and site-specific DNA recombination in transgenic mice. Proceedings of the National Academy of Sciences USA89: 6861–6865.
30.
PontiusBWLottWB & von HippelPH (1997). Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism. Proceedings of the National Academy of Sciences USA94: 2290–2294.
31.
RabbiMFSaifuddinMGuDSKagnoffMF & RoebuckKA (1997). U5 region of the human immunodeficiency virus type 1 long terminal repeat contains TRE-like cAMP-responsive elements that bind both AP-1 and CREB/ATF proteins. Virology233: 235–245.
32.
RoebuckKAGuDS & KagnoffMF (1996). Activating protein-1 cooperates with phorbol ester activation signals to increase HIV-1 expression. AIDS10: 819–826.
33.
SakaiAHirabayasiYAizawaSTanakaMIdaS & OkaS (1999). Investigation of a new p24 antigen detection system by the chemi-luminescence-enzyme-immuno-assay. Journal of the Japanse Association for Infectious Diseases73: 205–212.
34.
SauerB (1987). Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Molecular and Cellular Biology7: 2087–2096.
35.
SauerB & HendersonN (1988). Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proceedings of the National Academy of Sciences USA85: 5166–5170.
36.
SauerB & HendersonN (1989). Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome. Nucleic Acids Research17: 147–161.
37.
SauerB & HendersonN (1990). Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase. New Biology2: 441–449.
38.
SauerB (1992). Identification of cryptic lox sites in the yeast genome by selection for Cre-mediated chromosome translocations that confer multiple drug resistance. Journal of Molecular Biology223: 911–928.
39.
SauerB (1993). Manipulation of transgenes by site-specific recombination: Use of Cre recombinase. Methods in Enzymology225: 890–900.
40.
SodroskiJGRosenCA & HaseltineWA (1984). Trans-acting transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected cells. Science225: 381–385.
41.
SymonsRH (1989). Self-cleavage of RNA in the replication of small pathogens of plants and animals. Trends in Biochemical Sciences14: 445–450.
42.
ThompsonJDAyersDFMalmstormTAMacKenzieTLGanousisLChowriraBMCoutureL & StinchcombDT (1995). Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter. Nucleic Acids Research23: 2259–2268.
43.
Van LintCAmellaCAEmilianiSJohnMJieT & VerdinE (1997). Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity. Journal of Virology71: 6113–6127.
44.
YuMPoeschlaEYamadaODegrandisPLeavittMCHeuschMYeesJK & Wong-StaalF (1995). A. Hampel, in vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1. Virology206: 381–386.
45.
ZhouCBahnerICLarsonGPZaiaJARossiJJ & KohnEB (1994). Inhibition of HIV-1 in human T-lymphocytes by retrovi-rally transduced anti-tat and rev hammerhead ribozymes. Gene149: 33–39.
46.
ZhouCBahnerIRossiJJ & KohnDB (1996). Expression of hammerhead ribozymes by retroviral vectors to inhibit HIV-1 replication: Comparison of RNA levels and viral inhibition. Antisense Nucleic Acid Drug Development6: 17–24.
47.
ZhouD-MZhangL-HKumarPKR & TairaK (1996). Ribozyme mechanism revisited: Evidence against direct coordination of a Mg2+ ion with the pro-R oxygen of the scissile phosphate in the transition state of a hammerhead ribozyme-catalyzed reaction. Journal of the American Chemical Society118: 8969–8970.
48.
ZhouD-MZhangL-H & TairaK (1997). Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5-oxy and 5-thio-substrates by a hammerhead ribozyme. Proceedings of the National Academy of Sciences USA94: 14343–14348.
49.
ZhouD-MHeQ-CZhouJ-M & TairaK (1998). Explanation by a putative triester-like mechanism for the thio effects and Mn2+ rescues in reactions catalyzed by a hammerhead ribozyme. FEBS Letters431: 154–160.
