摘要
单链可变区抗体(Single Chain Antibody Variable Region Fragments sFv)由完整抗体的可变区轻链(L_H)及可变区重链(V_H)组成，中间由一多肽链相连。近年来抗体工程技术的飞速发展使得这一具备完整抗体高特异性和结合性的最小结构单位，可通过适当的表达载体被导入靶细胞内的特定区域，并稳定、持续地表达目的基因，从而起到阻断或开放某缺陷基因的作用，故这一技术在当今肿瘤性疾病及病毒感染性疾病基因治疗中显示出广泛的应用前景。HIV-1为一具有包膜的逆转录病毒，含有2个拷贝的单链RNA(9800bp)。HIV-1借高亲合性的受体(CD_4)进入靶细胞，在宿主细胞酶的作用下脱去包膜并在病毒酶的作用下逆转录成cDNA进而整合于宿主细胞基因组，完成其生命周期。因此整合过程是其生命周期中不可缺少的关键步骤，阻断HIV-1整合酶将遏制HIV-1感染于早期阶段，从而起到治疗目的。据此，我们特构建了抗HIV-1整合酶的sFv(Anti-HIV-1 IN sFv)及带有核内定位信号的抗HIV-1整合酶的sFv(Anti-HIV-1 IN／Nu sFv)。
一、抗HIV-1整合酶sFv的功能鉴定
为探讨IN-sFv在HIV-1感染中的临床应用意义。作者对其进行了功能测试。首先以免疫染色法对该结构的细胞内表达及分布进行了观察。结果显示。转染有IN-sFv及IN／Nu-sFv的NIH3T3细胞分别于胞浆及胞核中有明确的sFv表达。然后对其进行了感染实验，以MOI=0.04-0.06的NL_(4-3)病毒株同时感染10~6个转染有IN-sFv的SupT1细胞及转染有CAT、HBVcore-sFv及抗HIV-1 Rev-sFv的SupT1对照组细胞。通过ELISA法测定培养细胞上清中的P~(24)蛋白含量证实IN-sFv阻断病毒复制的作用达
Single-chain Fv(sFv) are recombinant antibody fragments consisting of only the variable light chain (V_L) and variable heavy chain (V_H) domains covalently connected to one another by a polypeptide linker. Recent advances in antibody engineering have now allowed the genes encoding the sFv, which is probably the minimal structural domain that retains the specific and high-affinity binding properties of the parent antibody , to be stably expressed in precise intracellular locations inside mammalian cells. As a simple and effective new method to replace defective genes or add functional genes to specific cell types, sFv gene therapy is likely to have widespread impact as potential therapeutic agents on both tumor and infectious diseases.
HIV-1 is an enveloped retrovirus containing two copies of a single-stranded RNA genome. After binding to a high-affinity receptor on a cell surface, called the CD4 molecule, the virion penetrates the cell and is uncoated by cellular enzymes. Viral reverse transcriptase catalyzes the synthesis of a double-stranded DNA intermediate from the viral RNA. The DNA is transported to the nucleus by the viral proteins and integrated into the host cell's genome by viral integrase(IN) to complete its life cycle. As HIV-1 integrase plays a key role in the early stage of its life cycle, successfully blocking the integrase will halt the HIV-1 replication prior to integration. Based on these, we constructed sFv which binds to the HIV-1 integrase C-terminal domain.
引文
1. Kevy J A. Pathogenesis of human immunodeficiency virus infection. Microbial Rev, 1993; 57: 183-289.
2. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immunedeficiency syndrome. Science, 1983; 220: 868-871.
3. Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent detection and isolation of cytopathic retroviruses from patients with AIDS and at risk for AIDS. Science, 1984; 224: 500-503.
4. Levy JA, Hoffman AD, Kramer SM, et al. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science, 1984; 225: 840-842.
5. Schnittman S. M, M. C. Psallidopoulos H. C, Lane L. Thompson, et al. The reservoir for HIV-1 in human peripheral blood is a T cell that maintains expression of CD4. Science, 1989; 245: 305-308.
6. Koenig S, H. E. Gendelman, J. M. Orenstein, et al. Detection of AiDS virus in macrophages in brain tissue of AIDS patients with encephalopathy. Science, 1986; 233: 1089-1093.
7. Katz R. A, A.m. Skalka. The retroviral enzymes. Annu Rev Biochem, 1994; 63: 133-173.
8. Brown P. O, B. Bowerman,. H. E. Varmus, et al. Correct integration of retroviral DNA in vitro. Cell, 1987; 49: 347-356.
9. Elison V, H. Abrams, T. Roe, et al. Human immunodeficiency virus integration in a cell-free system. J. Virol, 1990; 64: 2711-2715.
10. Engelman A, K. Mizuuch, R. Craigie. HIV-1 DNA integration mechanism of viral DNA cleavage and DNA strand transfer. Cell, 1991; 67: 1211-1221.
11. Duan LX, Pomerantz J R. Intracellular antibodies for HIV-1 Crane therapy. Science and Medicine, 1996; May/June: 24-31.
12. Didier Trono. HIV accessory proteins: Leading roles for the supporting cast. Cell, 1995; 82: 189-192.
13. Craigie R, T. Fujiwara, F. Bushman. The IN protein of moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell, 1990; 62: 829-837
14. Farnet CM, WA. Haseltine. Determination of viral proteins present in the human immunodeficiency virus type I preintegration complex. J. Virol, 1991; 65: 1910-1915.
15. Katz RA, G. Merkel, J. Kulkosky, et al. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell, 1990; 63: 87-95.
16. Engelman A, AB. Hickman, R. Craigie. The core and carboxylterminal domains of the integrase protein of human immumodeficiency virus type 1 each contribute to nonspecific DNA binding. J. Virol, 1994; 68:5911-5917.
17. Kulkosky J, KS. Jones, RA. Katz, et al. Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral retrotransposon integrases and bacterial insertion sequence transposases. Mol. Cell. Biol, 1992; 12: 2331-2338.
18. Andrake MD, AM. Skalka. Multimerization determinants reside in both the catalytic core and C-Terminus of avian sarcoma virus integrase. J. Biol, 1995; 270: 29299-29306.
19. Jenkins TN, A. Engeiman, R. Ghidando, et al. A soluble active mutant of HIV-1 integrase. J. Biol. Chem, 1996; 271: 7712-7713.
20. Kulkosky J, AM. Skalka. Molecular mechanism of retroviral DNA integration. Pharmacol.Ther, 1994; 61: 185-203.
21. pryciak PM, HE. Varmus. Nucle4osomes, DNA-binding proteins and DNA sequence modulate retroviral integration target site selection. Cell, 1992; 69: 769-780.
22. Sherman PA, ML. Dickson, JA. Fyfe. Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions, J. Virol, 1992; 66: 3593-3601.
23. Greene WC. The molecular biology of human immunodeficiency virus type 1 infection. N. Engl. J. Med, 1991; 324: 307-317
24. Cullen BR. Human immunodeficiency virus as a prototypic complex retrovirus. J. Virol, 1991; 65: 1053-1056.
25. Hirsch MS, RT. D'Aquila. Therapy for human immunodeficiency virus infection. N. Engl. J. Med, 1993; 328: 1686-1695.
26. Pomerantz RJ D. Trono. Genetic therapies for human immunodeficiency virus infections: Promise for the future. AIDS, 1995; 9:985-993.
27. St. Clair MH, JL. Martin, G. Tudor-Williams, et al. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science, 1991; 253: 1557-1559.
28. Erice A, HH. Balfour. Resistance of HIV-1 to anti-retroviral agents: a review. Clin. Infect. Dis, 1994; 18: 149-156.
29. Dropulic B, K-T. Jeang. Gene therapy for human immunodeficiency virus infection: genetic antiviral strategies and targets for intervention. Hum. Geone Ther, 1994; 5: 927-937.
30. Duan L-X, O. Bagasra, MA Laughlin, et al. Protent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc. Natl. Acad. Sci USA, 1994; 91: 5057-5079.
31. Marasco WA, WA Haseltine, I.S-Y. Chen. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gpl20 single -charn antibody. Proc. Natl. Acad. Sci. USA, 1993; 90: 7889-7893.
32. Feramisco JR, Clark R, Wong G, et al. Transient reversion of ras oncogene-induced cell transformation by antibodies specific for amino acid 12 of ras protein. Nature(Lond.), 1985; 314: 639-642
33. Morgan DO, Ross R. Analysis ofintracellular protein function by antibody injection. Immunol Today, 1988; 9: 94-86.
34. Richardson JH, Sodroski JG, Waldmann TA, et al. Phenotypic Knockout of the highaffinity human interleukin-2 receptor by intracellular single-chain antibodies against the subunit of the receptor. Proc Natl Acad Sci USA, 1995; 92: 3137-3141.
35. Deshane J. Targeted tumor killing via an intracellular antibody against c-erbB-2. J Clin Invest, 1995; 96: 2980-2989.
36. Teillaud J-L. Intracelllular targeting by anti-p21-ras sFv: a model for-anti tumor therapy. Exploring and Exploiting Antibody and Ig Superfamily combining Sites. Keystone Symposia, New Mexico, 1996; February P46.
37. Montano X, Jimenez A. Intracellular expression of the monoclonal anti-ras antibody Y13-259 blocks the transforming activity of ras oncogerms. Cell Growth and Differentiation, 1995; 6: 597-605.
38. Maciejwski Jp. Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro. Nat Mag, 1995; 1: 667-673.
39. Poznansky MC. Intracellular antibodies against Tat and gp120 inhibit the replication of HIV-1 in T-cells from HIV infected individuals. Xth Internatinal AIDS Conference. Vancouver, 1996(Abstract).
40. Jones SD. Gene therapy for HIV using intracellular anti-bodies. J Cell Bio, 1995; 21A: 395.
41. Pnina LE, L-X Duan, H-Z Zhang et al. 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. Virology,1996; 70: 8821-8832.
42. Bendeerrrrr DB, J. Kukosky, A. M. Skalka. Monoclonal antibodies against HIV type 1 integrase: clues to molecular structure. AIDS Res. Hum. Retroviruses, 1994; 10: 1105-1115.
43. Miller AD, GJ. Roseman. Improved retroviral vectors for germ transfer and expression. Bio Techniques, 1989; 7: 980-990.
44. Poiesz BJ, GD. Ehrlich, BC. Byrne, et al. In L. M. de la Maza and E. M. Peterson(ed). Medical virology 1990; 9: 47-75, Plenum Press, Mew York.
45. Ausubel FM, R. Brent, RE. Kingston, et al. Current protocols in molecular biology. 1994; Vol. 1. John Wiley &Sons, Inc., New York.
46. Gallay P, S. Swingler, J.Song, et al. HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell 1995; 83: 569-576.
47. Duan L-X, RJ. Pomerantz. Intracellular antibodies for HIV-1 Gene therapy. Sci. Med, 1996; 3: 24-33.
48. Bukrinsky M, N Sharova, M Stevenson. Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex. J. Virol, 1989; 67: 6863-6865.
49. Pauza CD, J Galindo. Persistent human immunodeficiency virus type 1 infection of monoblastoid cells leads to accumulation, of self-integrated viral DNA and to production of defective virions. J. Virol, 1989; 63: 3700-3707.
50. Farnet CM, Haseltine WA. Circularization of human immunodeficiency virus type 1 DNA in vitro. J. Virol, 1991; 65: 6942-6952.
51. Engelman A, G. Englund, JM. Orenstein, et al. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J. Virol, 1995; 69: 2729—2736.
52. Lutzke P, R A C Vink, R H A Plasterk. Characterization of the minmal DNA-binding domains of the HIV integrase protein. Nucleic Acids Res. 1994; 22: 4125-4131.
53. Cannon P M, E D. Byles, S M Kingsman, et el. Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replicatrion. J. Virol, 1996; 70: 651-657.
54. Desrosiers TC. The simian immunodeficiency viruses. Annu. Rev. Immune, 1989; 8: 557-558.
55. Fauci A, A. Macher, D. Longo, et al. Acquired immunodeficiency syndrome: epidemiologic, clinical, immuologic and therapeutic considerations. Ann. Intern. Med, 1984; 100: 92-106.
56. Clavel F, D. Guetard, F. Brun-Vezinet, et el. Isolation of a new human retrovirus from West African patients with AIDS. Science, 1986; 233: 343-346
57. Desrosiers RC, MD. Daniel, Y. Li. HIV related lentiviruses of nonhuman primates. AIDS Res. Hum. Retroviruses, 1989; 5: 465-473.
58. JavaherianK, JJ. Langlois, S. Schmidt, et al. The principal neutralization determinant of simian immunodeficiency virus differs from that of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA, 1992; 89: 1418-1422.
59. Johnston MI, Hoth DF, Present status and future prospects for HIV therapies. Science 1993; 260: 1286-1293.
60. Larder BA, Darby G, Richman DD. HIV with reduced sensitivity to Zidovudine(AZT) isolated during prolonged therapy. Science 1989; 243: 1731-1734.
61. Gao Q, Gu ZX, Parniak MA, et al. In vitro selection of viron of human immunodeficiency virus type 1 resistance to 3'-azido-3'deoxythymidine and 2',3'- dideoxyinosine. J. Virol, 1992; 66: 12-19.
62. Larder BA, Kemp SD. Mutiple mutations in HIV-1 reverse transcriptase confer high level resistance to Zidovudine(AZT). Science 1989; 246: 1155-1158.
63. Danos O, Mulligan RC. Safe and efficient generatiion of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci, USA, 1988; 85: 6460-6464.
64. Miller AD, Muttlmore S. Redesign nf retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell Biol, 1986; 6: 2895-2902.
65. Miller AR, Skotzko MJ, Phoades K, et al. Simultaneous use of two retroviral vectors in human gene marking trials: feasibility and potential applications. Hum. Gene Ther. 1992; 3: 619-624.
66. Muenchau DD. Freeman SM, Cornetta K et al. Analysis of retroviral packaging lines for generation of replication-competent virus. Virology, 1990; 176: 262-265.
67. Biocca S, Pierandrei-Amaldi P, Cattaneo A. Intracellular expression of anti-p21~(ras) single chain Fv fragments inhibits meiotic maturation of xenopus ocytes. Biochem Biophy Res Comm, 1993; 197: 422-427.
68. Biocca S, Pierandrei-amaldi P, Campioni N, et al. Intracellular immunization with cytosolic recombinant antibodies. Bio/Technology, 1994; 12: 396-399.
69. Werge TM, Baldari CT, Telford Jl. Intracellular single chain Fv antibody inhibits Ras activity in T-cell antigen receptor stimulated Jurkat cells. FEBS Lett, 1994; 351: 393-396.
70. Poul M-A, Teyssier C Lefranc MP. Inhibition of cyclin D1 incigene with intracellular single chain antibodies. Exploring and Exploring Antibody and Ig Superfamily Combining Site. Keystone Symposia, Mew Mexico, 22-28 February 1996.
71. Braun SE, Pan D, EL Aronovich, et al. Preclinical studues of lymphocyte gene therapy for mild hunter syndrome. Hum Gene Ther, 1996; 7283-290.
72. Burton DR. Large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries nf asymptomatic sero-positive individuals. Proc Narl Acad Sci USA 1991; 88: 10134-10137.
73. Barbas CF. Recombinant human Fab fragments neutralize human type limmunodeficiency virus in vitro. Proc Marl Acad Sci USA, 1992; 89: 9339-9343.
74. Zebedee SL. Human combinatorial antibody libraries to hepatitis B surface antigen. Proc Nail Acad Sci USA 1992; 89: 3175-3179.
75. Duan L. Potent inhibition of human immunodeficiency virus type lreplication bu an intracellular anti-Rev single chain antibody. Proc Nail Acad Sci USA, 1994; 91: 5075-5079.
76. Duan L, Zhu M, Bagasra O, et al. Intracellular immunization against HIV-1 infechon of human T lymphocytes: utility of anti-Rev single chain variable fragments. Hum Gene Ther, 1995; 16: 1561-1573.
77. Marasco WA. Intrabodies: turning the humeral immune system outside in for intracellular immunization. Gene Ther, 1997; 4: 11-15.
78. Danes O. In "Methods in Molecular Virology". Vol. 8. "Practical Molecular Virology: Viral Vectors for Gene Expression" (M. Collins. Ed). pp.17-27. Humana Press Inc. Clifton, NJ, 1991.
79. Joshi A, Jeang K-T: Reduction in growth temperature minimizes instablity of large plasmids containing HIV-1 proviral genomes. BioTechniques 1993; 14: 883-886.
80. Cosset FL, Flamant GA, Valsesia DAR, et al. Use of helper cells with two host ranges to generate high titer retroviral vectors. Virology, 1993; 193: 385-395.
81. Donahue RE, Kessler SW, Bodine D, et al. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated genetransfer. J Exp Med, 1992; 176: 1125-1135.
82. Haapala DK, Tobey WG, Oroszlan SD, et al. Isolation from cats of an endogenous type C virus with a novel envelope glycoprotein. J. Virol, 1985; 53: 827-833.
83. Runstadler PW, Uoung MW. GMP production of biopharmaceuticals using high density, fluidized-bed cell culture technology. In Animal Cell Culture and Production of Biologicals. S. Sasaki and K. Lkura. eds. (Kluwer aAcademic Publishers Dordrecht)pp. 103-119.
84. Kotani H, PB. NewtonIII, SY Zhang, et al. Improved methods of retroviral vector transduction and production for gene therapy. Hum Gene Ther, 1994; 5: 19-28.
85. Smith KT, AJ Shepherd, JE Boyd, et al. Gene delivery systems for use in gane therapy: an overview of quality assurance and safety issues. Gene Ther, 1996; 3: 190-200.
86. Center for biologics evaluation and research. The Food and Drug Administration. Points to Consider in Human Somatic Cell Therapy and Gene Therapy. Hum. Gene Ther, 1991; 2: 251-256.
87. Anderson WF, McGarrity GJ, Moen RC. Report to the NIH Recombinant DNA Advisory Committee on Murine Teplication-Competent Retrovirus (RCR) Assays. Hum Gene Ther, 1993; 4: 311-321.
1. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acpuired immune deficiency syndrome(AIDS). Science 1983; 220: 868-871.
2. Gallo RC, Salahuddin SZ, Popovic M, Shearer GM, et al. Frepuint detection and isolation of cytopathic retroviruses(HTLV-Ⅲ) from patients with AIDS and at risk for AIDS. Science 1984; 224: 500-503.
3. LevY JA, Hoffman AD, Kramer SN, et al. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 1984; 225: 840-842.
4. Ratner L, Haseltine W, Patarca R, et al. Complete nucleotide sequence of the AIDSvirus, HTLV-Ⅲ. Nature 1985; 313: 277-285.
5. Muesing MA, Smith DH, Cabradilla CD, et al. Nucleic acid structure and expression of the human AIDS/Lymphadenopathy retrovirus. Nature 1985; 313: 450-458.
6. Clavel F, Guetard D, Brun-Veziner F, et al. Isolation of amew human retrovirus from West African patients with AIDS. Science 1986; 233: 343-346.
7. Guyader M. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature 1987; 326: 662-669.
8. Starcich B, Ratner L, Josephs SF, et al. Characterization of long terminal repeat sequences of HTLV-Ⅲ. Science 1985; 227: 538-540.
9. Gaymor R. Cellular transcription factors involved in the regulation of HIV-1 gene expression AIDS 1992; 6: 347-363
10. Pearson L, Garcia J, Wu G, et al. Atransdominant tat mutant that inhibits tat-induced gene expression from the human immunodeficiency virus long terminal repeat. Proc. Natl. Acad. Sci. USA 1990; 87: 5079-5083.
11. Sullenger BA, Gallardo GF, Ungers GE, et al. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell 1990; 63: 601-608.
12. Tilley LS, Brown PH, Cullen BR. Does the human immunodeficiency virus Tat transactivator contain a discrete activation domain? Virology 1990; 178: 560-567.
13. Jacks T, Varmus HE. Expression of the Rous Sarcoma virus pol Gene by ribosomal frameshifting. Science, 1985; 230: 1237-1242
14. Tacks T, Power MD, Masiarz FR, et al. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature, 1988; 331:: 280-285
15. Jacks T, Madhonl HD, Masiarz FR, et al. Signals for ribosomal frameshifting in the Rous Sarcoma virus gag-pol region. Cell 1988; 55: 447-458
16. Veronese FDM, Coneland TD, Devieo AL, et al. Charaterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-Ⅲ/LAV Science, 1986; 231: 1289-1293
17. Mitsuya H, Yarchoan R, Broder S. Molecular targets for AIDS therapy. Science 1990; 249: 1533-1544.
18. Erice A, Balfour HH. Resistance of HIV-1 to anti-retroviral agents: a review. Clin Infect Dis, 1994; 18: 149-156
19. Preston BD, Poiesz BJ, Loeb LA, et al. Fidelity of HIV-1 reverse transcriptase. Science, 1988; 243: 1168-1171
20. Donehower LA, Varmus HE. A mutant murine leukemia virus with a single missense codon in plo is defective in a function affecting integration. Proc. Natl. Acad. Sci. USA, 1984; 81: 6461-6465.
21. Panganiban AH, Temin HM. The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locusProc. Natl. Acad. Sci. USA 1984; 81: 7885-7887.
22. Schwartzberg D, Coliceli J, Goff SP, et al. Construction and analysis of deletion mutations in the pol gene of Moloney Murine Leukemia virus: A new viral function required for productive infection. Cell 1984; 37: 1043-1052
23. Engelman A, Hickman AB, and Craigie R. The core and carboxyl-terminal domains of the integerse protein of human immunodeficienvy virus type 1 each contribute to nonspecific DNA binding. J. virol. 1994; 68: 5911-5917.
24. Kulkosky J, Jones KS, Katz RA, et al. Residues critical for trtroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and baterial insertion sequence transposases. Mol. Cell. Biol. 1992; 12: 2331-2338.
25. Andrake MD, and Skalka AM. Multimerization determinants reside in oth the catalytic core and C-terminus of avian sarcoma virus integrse. J. Biol 1995; 270: 29299-29306.
26. Jenkins TM, Engelman A Ghirlando R, et al. A soluble active mutant of HIV-1 integrase. J. Biol. Chem. 1996; 271: 7712-7713
27. Craigie K, Fujiwara T, and Bushman F. The IN protein of Moloney routine leukemia virus processes the viral DNA ends and. accomplishes their integration in vitro. Cell 1990; 62: 829-837.
28. Farnet CM, and Haseltine WA. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J. Virol 1991; 65: 1910-1915.
29. Katz TA, Merkel G, Kulkosky J, et al. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell 1990; 63: 87-95.
30. Engelman A, Mizuuch K, and Craigie R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell 1991; 67: 1211-1221.
31. Kulknsky J and Skalka AM. Molecular mechanism of retroviral DNA integration. Pharmacol. Ther 1994; 61: 185-203.
32. Pryciak PM, and Varmus HE. Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection. Cell 1992; 69: 769-780.
33. Sherman PA, Dickson ML, and Fyfe JA. Human immunodficiency virus type lintegration protein: DNA sequence requirements for cleaving and joining reactions. J. Virol. 1992; 66: 3593-3601.
34. Engelman A, Hickman AB, and Craigie R. The core and carboxyl-terminal domains of the integerse protein of human immunodeficienvy virus type 1 each contribute to nonspecific DNA binding. J. virol. 1994; 68: 5911-5917.
35. Kulkosky J, Jones KS, Katz RA, et al. Residues critical for trtroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and baterial insertion sequence transposases. Mol. Cell. Biol. 1992; 12: 2331-2338.
36. Andrake MD, and Skalka AM. Multimerization determinants reside in the catalytic core and C-terminus of avian sarcoma virus integrase. J. Biol 1995; 270: 29299-29306.
37. Jenkins TM, Engelman A Ghirlando R, et al. A soluble active mutant of HIV-1 integrase. J. Biol. Chem. 1996; 271: 7712-7713.
38. McDongal JS, Kennedy MS, Sligh JM, et al. Binding of HTLV-Ⅲ/LAV to T4+ T cells by a complex of the 110K virus protein and T4 molecule. Science, 1986; 231: 382-385.
39. Willey RL, Rutledge RA, Dias S, et al. Identification of conserved and divergent domains within the envolope gone of the acquired immunodeficiency syndrome retrovirus. Proc Natl Acad Sci USA 1986; 83: 5038-5042
40. Desai SM, Kalyanaraman VS, Casey JM, et al. Molecular cloning and primary nucleotide seqeuence analysis of a distinct human immunodeficiency virus isolate reveal significant divergence in its genomic sequences. Proc Natl Acad Sci USA 1986; 83: 8380～8384
41. Frankel AD, Chen l, Cotter RJ, et al. tat protein from human immunodeficiency virus forms a metal-linker dimer. Science 1988; 240: 70-73
42. Frankel AD, Chen L, Cotter RJ, et al. Dimerization of the tat protein from human immunodeficiency virus: A cysteine-rich peptide minics the normal metal-linker dimer interface. Proc. Natl. Acad. Sci. USA, 1988; 85: 6297～6301
43. Flossie WS, Salahuddin SZ, Popovic M, et al. Trans-acting transcriptional regulation of human T cell leukemia virus type Ⅲ long terminal repeat. Science, 1985; 227: 171～173
44. Dingwall C, Ernberg I, Gait MJ, et al. Human immunodeficiency virus 1 tat protein binds trans-aetivation-responsive region(TAR) RNA in vitro. Proc. Natl. Acad. Sci. USA, 1989; 86: 6925～6929
45. Gerke V, Steitz JA. Aprotein associated with small nuclear ribonuceoprotein psrticles recognizes the 3' splice site of premessenger RNA. cell, 1986; 46: 973～984
46. Frankel AD, Pabo Co. Cellular uptake of the Tat protein from human immunodeficiency vires. Cell 1988; 55: 1189～1193
47. Chang DD, Sharp PA. Messenger TNA transport and HIV rev regulation. Science, 1990; 249: 614～615
48. Felber BK, Margarita HC, Cladaras C et al. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc. Nail. Acad. Sci. USA, 1989; 86: 1495～1499
49. Malin MH, Hauber J, Le SY, et al. The HIV-1 rev trans-activator acts through a structured target sequence to activate muclear export of unspliced viral mRNA. Nature, 1989; 338: 254～257
50. Luciw PA, Cecilia CM, Levy, JA. Mutational analysis of the human immunodeficiency virus: the orf-Bregion down-regulates virus replication. Proc. Natl. Acad. Sci. USA, 1987; 84: 1434～1438
51. Terwilligen EG, Coheen EA, Lu YC, et al. Functional role of human immunodeficiency virus type 1 vpu. Proc. Nail. Acad. Sci. USA, 1989; 86: 5163～5167
52. Fisher AG, Ensoli B, IvanoffL, et al. The sor gene of HIV-1 is required for efficient virus transmission in vitro. Science, 1987; 237: 888～893
53. Bednarik DP, Mosca JD, Raj NBK, et al. Inhibition of human immunodeficiency virus(HIV) replication by HIV-trans-activated α2-interferon. Proc. Natl. Acad. Sci. USA, 1989; 86: 4958～4962
54. Sullenger BA, Gallardo HF, Ungers GE, et al. Over expression of TAR sequences renders cells resistant to human immunodeficiency virus replication, Cell 1990; 63: 601～608
55. Lisziewicz J, Sun D, Smythe J, 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
56. Lee S-W, Gallardo GF, Giboa E,et al. Inhibition of human immunodeficiency virus type 1 in human Tcells by a potent Rev response element decoy consisting of the 13 nucleotide minimal Rev-binging domain. J Virol, 1994; 68:8254～8264
57. Buck HM, Koole LH, Genderen MHP, et al.Phosphatemethlated DNA aimed at HIV-1 DNA loops and integrated DNA inhibites viral infectivity. Science,1990; 248: 208～212
58. Lisziewicz J, Sun D, Klotman M, et al.Specific inhibition of human immunodificiency virus type 1 replication by antisence oligonucleotides: an in vitro model for treatmaent. Proc Natl. Acad. Sci. USA, 1992; 89: 11209～11213
59. Buchschacher GL. Molecular targets of gene transfer therapy for HIV infection. JAMA,1993; 269:2880～2886
60. Sarver N, Cantin EM, Chang PS, et al. Tibozymes as potential anti-HIV-1 therapeutic agents. Science, 1989; 247:1222～1225
61. Yu M, Ojwang J, Yamada O, et al. A hairpin ribozyme inhibits expression og diverse strains of human immunodeficiency virus type 1. Proc. Nail. Acad. Sci. USA, 1993; 90:6340～6344
62. Malim MH, Freimuth WW, Liu J, et al. Stable expression of transdominant rev protein in human T cells inhibits human immunodeeficieney virus replication. J Exp Med, 1992; 176:1197～1201
63. Modesti N, Garcia J, Debouck C, et al. Trans-dominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression. The New Biologist, 1991; 3:759～768
64. Trono D, Feinberg MB, Baltimore D. HIV-1 Gag mutants dominantly interfere with the replication of the wild-typo virus. Cell, 1989; 59:113～120
65. Buchschacher GL Jr, Freed EO, Panganiban AT. Cells induced to express a human immunodeficiency virus type 1 envelope gene mutant inhibiit the spread of wild type virus. Hum Gene Therapy, 1992; 3:391～397
66. Duan L, Bagasra O, Laughlin ML, et al. Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc Nat Acad Sci USA, 1994; 91:5075～5079
67. Duan L Zhang H, Oakes JW,et al. Molecular and virological effects of intracellular anti-Kev single-chain variable fragments on the expression of various human immunodeficiency virus-1 strains. Hum Gene Therapy, 1994; 5:1315～1324
68. Marasco W A, Haseltine WA, Chen SY.Design, intracellular expression, and activity of a human anti-HIV-1 gp120 single-chain antibody. Proc Nat Acad Sci USA, 1993; 90:7889～7893
69. Buonocore L, Rose JK. Blockade of human immunodeficiency virus type 1 production in CD4+ T cells by an intracellular CD4 espressed under control of the viral long terminal repeat. Pro Nat Acad Sci USA, 1993; 90:2695～2699
70. Venkatesh LK, Arens MQ, Subramanian T, et al. Selective induction of toxicity to human cells expressing human immucideficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector. Proc Nail Acad Sci USA,1990; 87:8746～8750
71. Harrison GS, Maxwell F, Lontg CJ, et al. Activation of a diphtheria toxin A gene by expression of human immunodeficiency virus-1 Tat and Key proteins in transfected cells. Hum Gene Therapy, 1991; 2:53～60
72. Baltimore D. Intracellular immunization. Nature, 1988; 335:395～396
73. Jeang KR,Berkhout B. Kinetics of HIV-1 long terminal repeat trans-activation. J Biol Chem, 1992; 264:17891～17899
74. Pomerantz RJ, Trono D. Fenetic therapies for HIV infections:promise for the future. AIDS, 1995; 9:985～993
75. Burton DR, Pyati J, Koduri T, et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science, 1994; 266: 1024～1028