细胞内生成的抗整合酶单链抗体阻断HIV-1复制的实验研究
作者：张惠中 朱明华 段凌寻
单位：张惠中(第四军医大学唐都医院骨科,陕西西安710038)；朱明华(上海第二军医大学病理学教研室)；段凌寻(美国ThomasJefferson大学)
关键词：基因治疗；单链可变区抗体；I型人免疫缺陷病毒；整合酶
细胞与分子免疫学杂志000101摘要：目的通过细胞内表达抗HIV-1整合酶单链抗体(IN-sFv)基因，阻断病毒基因组与宿主细胞基因组的整合，进而抑制病毒复制，探讨该基因载体在HIV-1感染基因治疗中应用的意义。方法用带有IN-sFv基因的逆转录病毒表达载体pSLXCMV/IN-sFv转染PA317包装细胞，并用包装后含有目的基因的逆转录病毒转导SupT1细胞及外周血单个核细胞(PBMC)。以HIV-1NL4-3病毒株感染表达IN-sFv的SupT1及PBMC，用ELISA方法测定病毒感染细胞后不同时间培养上清中HIV-1P24蛋白的含量，以监测病毒复制的水平；用半定量巢式PCR扩增病毒感染后不同时间点细胞内HIV-1整合前体中的环状病毒DNA，以明确病毒进入细胞后的整合状态。结果IN-sFv在SupT1及PBMC两种细胞中，均可明显地抑制病毒的复制。环状病毒DNA在表达IN-sFv的SupT1细胞中早于对照细胞8～10h出现。结论细胞内表达的抗HIV-1整合酶单链抗体，可显著抑制病毒的整合，从而阻断细胞内病毒的复制。该结果为开拓HIV-1基因治疗的新领域奠定了基础。
, 百拇医药
中图号：R373.9 文献标识码：A
Inhibition of HIV-1 replication by anti-integrase single chain Fv antibody generated in situ
ZHANG Huizhong
(Department of Orthopeadics, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038,Shaanxi Province, China)
ZHU Minghua
(Department of Pathology, Second Military Medical University, Shanghai,China)
, 百拇医药
(Thomas Jefferson University, USA)
DUAN Lingxun
Abstract:Aim To evaluate the therapeutic effects of anti-HIV-1 integrase sFv(IN-sFv) on the inhibition of HIV-1 replication. Methods Challenge experiments, utilizing the highly cytopathic viral strain NL4-3, were conducted with SupT1 cells and peripheral blood mononuclear cells(PBMCs) stably transduced with anti-HIV-1 IN-sFv gene. The spread of HIV-1 in the cultures was determined by quantitating the levels of HIV-1 p24 antigen released into the culture medium. To further confirm the mechanism of HIV-1 replication inhibition, semiquantitative 2-LTR PCR with nested primers was performed to amplify HIV-1 circled 2-LTR DNA at different time points after the anti-HIV-1 IN-sFv transduced and non-transduced SupT1 cells were infected with NL4-3 virus at a high MOIs of 2.0. Results The Challenge results showed approximately 95% to 98% inhibition of HIV-1 p24 antigen generation in the cells transduced with IN-sFv and IN-sFv-nls compared with the control cells. The 2-LTR PCR results demonstrated that the detectable levels of HIV-1 2-LTR DNA molecules occurred significantly earlier in cells expressing anti-HIV-1 IN-sFv than in the control cells. Conclusion The present data suggest that the IN-sFv can significantly inhibit HIV-1 replication via the blocking of HIV-1 integration and is therefore a promising therapeutic agent for gene therapy of the HIV-1 infection.
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Keywords: gene therapy; sFv; HIV-1; integrase
Integration of HIV-1 viral DNA into a chromosome of the infected host cells is the key step towards efficient virus replication, and this procedure is mediated by the virus-encoded enzyme integrase. As integrase plays a pivotal role in the HIV-1 infection and there are no effective anti-viral drugs targeted to the integrase, it is an attractive way to try the new method of blocking HIV-1 infection using intracellularly expressed sFv binding to the integrase(IN-sFv). The functional assay of the IN-sFv was conducted to explore the significance of this moities in the use of HIV-1 infection.
, 百拇医药
MATERIALS AND METHODS
Materials The recombinant retroviral vectors with one containing anti-HIV-1 integrase single chain variable fragment antibody gene(IN-sFv)which was amplified by RT-PCR from anti-intergrase monoclonal antibody producing hybridoma cells and another containing IN-sFv with Tat Nuclear Localization Signal gene were con structed by Dr. Duan Lingxun at Thomas Jefferson University using retroviral vector pSLXCMV which encoded a neomycin phosphate transferase gene as report gene. The recombinant retroviral vectors were named pSLXCMV/IN-sFv and pSLXCMV/IN-sFv-nls.
, 百拇医药
Methods
Cell cultures The packaging cells used in this study for the recombinant retroviral vector packaging to form an infection competent and replication deficiency virion were PA317 cells, and the NIH3T3TK- cells were used in the titration of packaged virion titer. Both cells were maintained in the 100 mL/L fetal bovin serum DMEM medium with 4.5 g/L glucose. SupT1 cells cultured in the RPMI-1640 medium suppliment with 100 mL/L fetal bovine serum were HIV-1 susceptible human lymphoma cells.
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Production and titration of recombinant retroviruses
Recombinant murine leukemia virus-based retroviruses were produced by transfection of PA317 packaging cells with 20 μ g recombinant retrovirus vector plasmids pSLXCMV/IN-sFv and pSLXCMV/IN-sFv-nls using standard calcium phosphate transfection method respectively for 14 hours and the medium was changed with fresh complete DMEM to continue to culture for another 24 h. 650 mg/L G418 were added into the medium and continued to maintain the cells until G418 resistant colonies formed. The colonies of PA317/IN- sFv and PA317/IN-sFv-nls cells were picked up and cultured with fresh complete DMED medium till the cells reached 90% - 100% confluence. The supernatants of cultured PA317/IN-sFv and PA317/IN-sFv-nls cells were collected and serially diluted for the use of titration of recombinant virus titer by infecting NIH3T3 cells followed by selecting culture with 650 mg/L G418 containing DMEM medium as described above.The colonies NIH3T3/IN-sFv cells were stained with Crystalblue and the colony forming units(cfu) were calculated. The highest cfus(2.8× 106)producing PA317 cells transduced with pSLXCMV/IN-sFv and pSLXCMV/IN-sFv-nls were used to create recombinat retrovirus containing supernatant.
, 百拇医药
Transduction of SupT1 and PBMCs ① For transduction of SupT1 cells, 5mL of G418 free supernatant containing virions produced by transfected PA317 cells was used to infect 1× 106 target cells for 24- 48 hours. Cells were then maintained under G418 selection condition for 2 weeks and followed for another 2 weeks culturing with G418 free medium before cells were used for HIV-1 infection assay. ② Isolation and transduction of PBMCs. Human PBMCs purified from buffy coats of HIV-1 seronegative individuals were stimulated with phytohemagglutinin(5 mg/L) and interleukin-2(2× 105U/L) for 3 d; then 106 PBMC were cultured with 10 mL of supernatant from the indicated sFv transfected PA317 cultures for 3 d, with daily replacement with fresh supernatant. The transduced PBMCs were then challenged with HIV-1 at the fourth day.
, 百拇医药
IN-sFv functional assay Parental SupT1 and PBMC cells alone and cells transduced with anti-IN sFvs, CAT, and the anti-HBVcore-sFv were incubated with infectious HIV-1 NL4-3 virions at multiplicities of infection(MOIs) of 0.04- 0.06 for 4 hours. The cells were washed four times with PBS solution and then maintained in growth medium. Every 3 d, cells were split 1∶ 2 to maintain a cell density of approximately 109/L and the culture supernatants were collected for HIV-1 p24 antigen analyses with ELISA method(Dupont Inc Kit).
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PCR amplification of HIV-1 2-LTR DNA with nested primers HIV-1 NL4-3 virus at a high MOI of 2.0 was used to infect nontransduced SupT1 cells and SupT1 cells which were transduced with anti-IN sFv and anti-IN-sFv-nls for 4 hours. Cells were then washed with PBS and cultured for different time periods. At 4,6,8 and 16 h after HIV-1 infection, cells were harvested by low-speed centrifugation and washed with PBS. Total cellular DNA was prepared by a quick lysis method. HIV-1 infection was cofirmed by assaying for the synthesis of viral DNA by PCR with the SK38-SK39 primer pair, which is located in the gag region of the HIV-1 genome. To amplify viral 2-LTR-DNA moieties, a nested PCR was developed with two primer pairs:M667-U32 and U5-2LTR-U3-2LTR. The first PCR, performed with primer pair M667-U32, was for 29 cycles in a 25 μ L volume with reaction conditions as follows: 5 min at 94℃ (for denaturation), 94℃ for 30 s, 58℃ for 30 s, 72℃ for 40 s. From the first PCR reaction mixture, 5 μ L of PCR product was further amplified with the U3-2LTR and U5-2LTR primer pair for 25 cycles under the same amplification conditions as were used for the first PCR round. The PCR products were separated on 15 g/L agarose gels, transferred onto membranes,and hybridized with a 32P-5′ -end-labeled oligonucleotid(AA50)probe specific for the U5 sequence and a 32P-5′ end-labeled SK19 probe specific for gag sequences.The 2-LTR DNA plasmid was used in this PCR reaction as a positive control. The sequences of the primers and probes used in this experiment were as follows:
, 百拇医药
M667:5′ -GGCTAACTAGGGAACCCACTG-3′ ;
U32: 5′ -GGCAAA AAGCAGC TG CTT-3′ ;
U5: 5′ -GAGATCCCTCAGACCCTTTTAG-3′ ;
U3: 5′ -GTAAGTGGATATCTGATCCCTG-3′ ;
SK38:5′ -ATAATCCACCTATCCCAGTAGGAGAAAT-3′ ;
SK39:5′ -TTTG GTC CT TGTCTTATGTCCAGAATGC-3′ ;
AA55:5′ -GCTAAGGATTTTCCACACTGA-3′ ;
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SK19:5′ -ATCCTGGGATTAAATAAAATAGTAAGAATGTATAG-
CCCTAC-3′ .
Immunoflorensence staining for IN-sFv expression
The intracellularly expressed IN-sFv proteins within the pSLXCMV/IN-sFv and pSLXCMV/IN-sFv-nls transduced SupT1 and PBMC cells were determined by indirect immunofluorescence assays. After the cell slides were prepared with CytoproTM machine(VIESCOR Inc and Methods manual), cells were then fixed in 35 g/L formaldehyde at room temperature for 10 min followed by treatment with a 1 g/L Nonidet P-40-PBS solution for 10 min; they were then washed twice with PBS. Cells were incubated with a 1∶ 200 dilution of goat anti-mouse polyclonal IgG Fab fragment and a 1∶ 500 dilution of FluoroLink-Cy2-labeled donkey anti-goat IgG (Amersham Life Science) for 2 h at 37℃ . After having been washed five times in PBS, cells were mounted and analyzed by epifluorescence microscopy.
, 百拇医药
RESULTS
1IN-sFv functional assay IN sFv-transduced SupT1 cells, CAT-,anti-Rev sFv-and anti-HBV core protein sFv-transduced SupT1 cells, and parental nontransduced SupT1 cells were infected with HIV-1 NL4-3 of the same MOI at 0.04～ 0.06. Syncytia and cell death and high levels of HIV-1 p24 antigen in the supernatants of nontransduced and CAT,anti-HBV core sFv transduced cells were observed at early time points after infection(10 d- 12 d), while there were only very low levels of HIV-1 p24 antigen in the supernatant of SupT1 cells transduced with IN sFv and few syncytia at the 30th- 34th day after infection(Figs 1,2,3).
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Fig 1 P24 level in the supernatant of NL4-3 challenged
SupT1 cells
Fig2 P24 level in the supernatant of NL4-3 challenged PBMCs
Fig 3 Results of NL4-3 virus challenged SupT1 cells(× 200)
A: Syncytia and dead cells of control SupT1 cells on 18th day;
, 百拇医药
B: SupT1 cells transduced with IN- sFv show no syncytia and dead cells on 18th day.
PCR amplification of HIV-1 2-LTR-DNA HIV-1 2-LTR-DNA PCR amplification of 4,6, 8,16 h infected nontransduced SupT1 cells and transduced with IN-sFv and IN-sFv-nls cells showed that 2-LTR-DNA were detectable 8- 10 h earlier in the SupT1 expressioning IN-sFv and IN-sFv-nls than in the nontransduced SupT1 cells(Fig4). HIV-1 gag gene PCR amplification demonstrated that the virus quantity among the wells for cell culture was the same.
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Fig 4 Results of Southern blot
A： 2-LTR-DNA Southern blot with AA55 probe;
B： gag gene Southern blot with SK19 probe.
(S: SupT1 cells; IN: IN-sFv transduced SupT1
cells; NL: IN-sFv-nls transduced SupT1 cells).
Immunofloresence staining The indirect immunofluorescence staining of intracellular expressed IN-sFv and IN-sFv-nls demonstrated that the anti-IN-sFv was mainly cytoplasmic staining and nucleoplasm exclusion while the anti-IN-sFv-nls was nuclear staining(Fig 5).
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Fig 5 Results of immunofluo-rescence staining
A:SupT1 cells transduced with IN-sFv show cytoplasm positive × 1000;
B:SupT1 cells transduced with IN-sFv-nls show nuclear positive × 200.
DISCUSSION
Our results demonstrated that intracellularly expressed anti-HIV-1 IN-sFv significantly inhibited the production of HIV-1 p24 protein both in SupT1 and PBMCs and the inhibition rate was approximately 95% - 98% compared with that control cells(Fig 1). There were no syncytia and cell death 30 days after virus infection either in SupT1 or in PBMCs. To determine whether the observed decreases in HIV-1 p24 antigen levels correlated with a specific intracellular inhibition of HIV-1 integrase activity or were merely secondary to other effects of intracellular expression of anti-IN sFv in T-lymphoid cells, levels of HIV-1 2-LTR-DNA were compared by semiquantitative PCR. The binding domain of the IN-sFv used in this experiment was the carboxy terminus of the HIV-1 integrase which had been shown to account for the nonspecific DNA affinity of the protein, and might be involved in target DNA interactions[3,4]. When a specific residue in the carboxy terminus of IN, Trp-235, was replaced by Ala, the ability of the HIV-1 provirus to replicate was abolished[5]. As shown in Fig. 4, the detectable levels of HIV-1 2-LTR-DNA molecules occurred 8- 10 hours earlier in cells expressing anti-IN-sFv or anti-IN-sFv-nls than in the control cells, which indicated that the virus cDNA accumulated and was ligated into a circled 2-LTR-DNA after it entered the infected cell nuclear due to the blocking of its integrase by intracellularly expressed anti-IN-sFv and it could not integrate into the infected cell genome any more[6,7}.
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The quantitative changes of 2-LTR-DNA at the same time point after infection was similar to those in the experiment results conducted with mutant integrase[8]. The difference between the two experiment results was that 2-LTR-DNA appeared at least 2 hours earlier in anti-IN-sFv-nls transduced cells in which the sFv was located in the nucleus, than in the cells transduced with anti-IN-sFv in which the sFv was located in the cytoplasmic compartment. The time difference suggests that HIV-1 IN may be initially sequestered by the cytoplasmic sFv ,delaying nuclear importation of the preintegration complex, where viral DNA ends are joined via nuclear ligase activity. Alternatively,IN-sFv-nls may facilitate its transport into the nuclear compartment by virtue of binding to the preintegration complex.
, 百拇医药
The results of this experiment further demonstrate that intracellularly expressed anti-HIV-1 IN-sFv can significantly inhibit virus replication via blocking the virus integrase and that the function of sFv is related to its intracellular localization. The sFv also serves as valuable reagents with which to explore further the combined target gene therapy methods in the blocking of HIV-1 replication.
Author： ZhangHZ,male， associate professor， Ph.D,M.D. Tangdu Hospital, Xinsi Road, Xi'an 710038, Shaanxi Province, China. Tel.(029)3577591
, 百拇医药
REFERENCE
〔1〕 Kulkosky J, Skalka AM. Molecular mechanism of retroviral DNA integration〔J〕 . Pharmacol Ther, 1994； 61(1- 2):185- 203.
〔2〕 Engelman A, Hickman AB, Craigie R. The core and carboxylterminal domains of the integrase protein of human immumodeficiency virus type 1 each contribute to nonspecific DNA binding〔J〕 . J Virol, 1994; 68(9):5911- 5917.
〔3〕 Lutzke RA, Vink C, Plasterk RH. Characterization of the minmal DNA-binding domains of the HIV integrase protein〔J〕 . Nucleic Acids Res, 1994; 22(20): 4125- 4131.
, 百拇医药
〔4〕 Cannon P M, Byles ED, Kingsman SM, et al. Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication〔J〕 . J Virol, 1996; 70(1): 651- 657.
〔5〕 Bukrinsky M, Sharova N, Stevenson M. Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex〔J〕 . J Virol, 1989; 67(11): 6863- 6865.
〔6〕 Pauza CD, Galindo J. 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〕 . J Virol, 1989; 63(9): 3700- 37007.
, 百拇医药
〔7〕 Farnet CM, Haseltine WA. Circularization of human immunodeficiency virus type 1 DNA in vitro〔J〕 . J Virol, 1991; 65(12): 6942- 6952.
〔8〕 Engelman A, Englund G, Orenstein JM,et al. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication〔J〕 . J Virol,1995; 69(5): 2729- 2736.
Received date:1999- 07- 13
revised date:1999- 09- 13, http://www.100md.com
单位：张惠中(第四军医大学唐都医院骨科,陕西西安710038)；朱明华(上海第二军医大学病理学教研室)；段凌寻(美国ThomasJefferson大学)
关键词：基因治疗；单链可变区抗体；I型人免疫缺陷病毒；整合酶
细胞与分子免疫学杂志000101摘要：目的通过细胞内表达抗HIV-1整合酶单链抗体(IN-sFv)基因，阻断病毒基因组与宿主细胞基因组的整合，进而抑制病毒复制，探讨该基因载体在HIV-1感染基因治疗中应用的意义。方法用带有IN-sFv基因的逆转录病毒表达载体pSLXCMV/IN-sFv转染PA317包装细胞，并用包装后含有目的基因的逆转录病毒转导SupT1细胞及外周血单个核细胞(PBMC)。以HIV-1NL4-3病毒株感染表达IN-sFv的SupT1及PBMC，用ELISA方法测定病毒感染细胞后不同时间培养上清中HIV-1P24蛋白的含量，以监测病毒复制的水平；用半定量巢式PCR扩增病毒感染后不同时间点细胞内HIV-1整合前体中的环状病毒DNA，以明确病毒进入细胞后的整合状态。结果IN-sFv在SupT1及PBMC两种细胞中，均可明显地抑制病毒的复制。环状病毒DNA在表达IN-sFv的SupT1细胞中早于对照细胞8～10h出现。结论细胞内表达的抗HIV-1整合酶单链抗体，可显著抑制病毒的整合，从而阻断细胞内病毒的复制。该结果为开拓HIV-1基因治疗的新领域奠定了基础。
, 百拇医药
中图号：R373.9 文献标识码：A
Inhibition of HIV-1 replication by anti-integrase single chain Fv antibody generated in situ
ZHANG Huizhong
(Department of Orthopeadics, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038,Shaanxi Province, China)
ZHU Minghua
(Department of Pathology, Second Military Medical University, Shanghai,China)
, 百拇医药
(Thomas Jefferson University, USA)
DUAN Lingxun
Abstract:Aim To evaluate the therapeutic effects of anti-HIV-1 integrase sFv(IN-sFv) on the inhibition of HIV-1 replication. Methods Challenge experiments, utilizing the highly cytopathic viral strain NL4-3, were conducted with SupT1 cells and peripheral blood mononuclear cells(PBMCs) stably transduced with anti-HIV-1 IN-sFv gene. The spread of HIV-1 in the cultures was determined by quantitating the levels of HIV-1 p24 antigen released into the culture medium. To further confirm the mechanism of HIV-1 replication inhibition, semiquantitative 2-LTR PCR with nested primers was performed to amplify HIV-1 circled 2-LTR DNA at different time points after the anti-HIV-1 IN-sFv transduced and non-transduced SupT1 cells were infected with NL4-3 virus at a high MOIs of 2.0. Results The Challenge results showed approximately 95% to 98% inhibition of HIV-1 p24 antigen generation in the cells transduced with IN-sFv and IN-sFv-nls compared with the control cells. The 2-LTR PCR results demonstrated that the detectable levels of HIV-1 2-LTR DNA molecules occurred significantly earlier in cells expressing anti-HIV-1 IN-sFv than in the control cells. Conclusion The present data suggest that the IN-sFv can significantly inhibit HIV-1 replication via the blocking of HIV-1 integration and is therefore a promising therapeutic agent for gene therapy of the HIV-1 infection.
, http://www.100md.com
Keywords: gene therapy; sFv; HIV-1; integrase
Integration of HIV-1 viral DNA into a chromosome of the infected host cells is the key step towards efficient virus replication, and this procedure is mediated by the virus-encoded enzyme integrase. As integrase plays a pivotal role in the HIV-1 infection and there are no effective anti-viral drugs targeted to the integrase, it is an attractive way to try the new method of blocking HIV-1 infection using intracellularly expressed sFv binding to the integrase(IN-sFv). The functional assay of the IN-sFv was conducted to explore the significance of this moities in the use of HIV-1 infection.
, 百拇医药
MATERIALS AND METHODS
Materials The recombinant retroviral vectors with one containing anti-HIV-1 integrase single chain variable fragment antibody gene(IN-sFv)which was amplified by RT-PCR from anti-intergrase monoclonal antibody producing hybridoma cells and another containing IN-sFv with Tat Nuclear Localization Signal gene were con structed by Dr. Duan Lingxun at Thomas Jefferson University using retroviral vector pSLXCMV which encoded a neomycin phosphate transferase gene as report gene. The recombinant retroviral vectors were named pSLXCMV/IN-sFv and pSLXCMV/IN-sFv-nls.
, 百拇医药
Methods
Cell cultures The packaging cells used in this study for the recombinant retroviral vector packaging to form an infection competent and replication deficiency virion were PA317 cells, and the NIH3T3TK- cells were used in the titration of packaged virion titer. Both cells were maintained in the 100 mL/L fetal bovin serum DMEM medium with 4.5 g/L glucose. SupT1 cells cultured in the RPMI-1640 medium suppliment with 100 mL/L fetal bovine serum were HIV-1 susceptible human lymphoma cells.
, http://www.100md.com
Production and titration of recombinant retroviruses
Recombinant murine leukemia virus-based retroviruses were produced by transfection of PA317 packaging cells with 20 μ g recombinant retrovirus vector plasmids pSLXCMV/IN-sFv and pSLXCMV/IN-sFv-nls using standard calcium phosphate transfection method respectively for 14 hours and the medium was changed with fresh complete DMEM to continue to culture for another 24 h. 650 mg/L G418 were added into the medium and continued to maintain the cells until G418 resistant colonies formed. The colonies of PA317/IN- sFv and PA317/IN-sFv-nls cells were picked up and cultured with fresh complete DMED medium till the cells reached 90% - 100% confluence. The supernatants of cultured PA317/IN-sFv and PA317/IN-sFv-nls cells were collected and serially diluted for the use of titration of recombinant virus titer by infecting NIH3T3 cells followed by selecting culture with 650 mg/L G418 containing DMEM medium as described above.The colonies NIH3T3/IN-sFv cells were stained with Crystalblue and the colony forming units(cfu) were calculated. The highest cfus(2.8× 106)producing PA317 cells transduced with pSLXCMV/IN-sFv and pSLXCMV/IN-sFv-nls were used to create recombinat retrovirus containing supernatant.
, 百拇医药
Transduction of SupT1 and PBMCs ① For transduction of SupT1 cells, 5mL of G418 free supernatant containing virions produced by transfected PA317 cells was used to infect 1× 106 target cells for 24- 48 hours. Cells were then maintained under G418 selection condition for 2 weeks and followed for another 2 weeks culturing with G418 free medium before cells were used for HIV-1 infection assay. ② Isolation and transduction of PBMCs. Human PBMCs purified from buffy coats of HIV-1 seronegative individuals were stimulated with phytohemagglutinin(5 mg/L) and interleukin-2(2× 105U/L) for 3 d; then 106 PBMC were cultured with 10 mL of supernatant from the indicated sFv transfected PA317 cultures for 3 d, with daily replacement with fresh supernatant. The transduced PBMCs were then challenged with HIV-1 at the fourth day.
, 百拇医药
IN-sFv functional assay Parental SupT1 and PBMC cells alone and cells transduced with anti-IN sFvs, CAT, and the anti-HBVcore-sFv were incubated with infectious HIV-1 NL4-3 virions at multiplicities of infection(MOIs) of 0.04- 0.06 for 4 hours. The cells were washed four times with PBS solution and then maintained in growth medium. Every 3 d, cells were split 1∶ 2 to maintain a cell density of approximately 109/L and the culture supernatants were collected for HIV-1 p24 antigen analyses with ELISA method(Dupont Inc Kit).
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PCR amplification of HIV-1 2-LTR DNA with nested primers HIV-1 NL4-3 virus at a high MOI of 2.0 was used to infect nontransduced SupT1 cells and SupT1 cells which were transduced with anti-IN sFv and anti-IN-sFv-nls for 4 hours. Cells were then washed with PBS and cultured for different time periods. At 4,6,8 and 16 h after HIV-1 infection, cells were harvested by low-speed centrifugation and washed with PBS. Total cellular DNA was prepared by a quick lysis method. HIV-1 infection was cofirmed by assaying for the synthesis of viral DNA by PCR with the SK38-SK39 primer pair, which is located in the gag region of the HIV-1 genome. To amplify viral 2-LTR-DNA moieties, a nested PCR was developed with two primer pairs:M667-U32 and U5-2LTR-U3-2LTR. The first PCR, performed with primer pair M667-U32, was for 29 cycles in a 25 μ L volume with reaction conditions as follows: 5 min at 94℃ (for denaturation), 94℃ for 30 s, 58℃ for 30 s, 72℃ for 40 s. From the first PCR reaction mixture, 5 μ L of PCR product was further amplified with the U3-2LTR and U5-2LTR primer pair for 25 cycles under the same amplification conditions as were used for the first PCR round. The PCR products were separated on 15 g/L agarose gels, transferred onto membranes,and hybridized with a 32P-5′ -end-labeled oligonucleotid(AA50)probe specific for the U5 sequence and a 32P-5′ end-labeled SK19 probe specific for gag sequences.The 2-LTR DNA plasmid was used in this PCR reaction as a positive control. The sequences of the primers and probes used in this experiment were as follows:
, 百拇医药
M667:5′ -GGCTAACTAGGGAACCCACTG-3′ ;
U32: 5′ -GGCAAA AAGCAGC TG CTT-3′ ;
U5: 5′ -GAGATCCCTCAGACCCTTTTAG-3′ ;
U3: 5′ -GTAAGTGGATATCTGATCCCTG-3′ ;
SK38:5′ -ATAATCCACCTATCCCAGTAGGAGAAAT-3′ ;
SK39:5′ -TTTG GTC CT TGTCTTATGTCCAGAATGC-3′ ;
AA55:5′ -GCTAAGGATTTTCCACACTGA-3′ ;
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SK19:5′ -ATCCTGGGATTAAATAAAATAGTAAGAATGTATAG-
CCCTAC-3′ .
Immunoflorensence staining for IN-sFv expression
The intracellularly expressed IN-sFv proteins within the pSLXCMV/IN-sFv and pSLXCMV/IN-sFv-nls transduced SupT1 and PBMC cells were determined by indirect immunofluorescence assays. After the cell slides were prepared with CytoproTM machine(VIESCOR Inc and Methods manual), cells were then fixed in 35 g/L formaldehyde at room temperature for 10 min followed by treatment with a 1 g/L Nonidet P-40-PBS solution for 10 min; they were then washed twice with PBS. Cells were incubated with a 1∶ 200 dilution of goat anti-mouse polyclonal IgG Fab fragment and a 1∶ 500 dilution of FluoroLink-Cy2-labeled donkey anti-goat IgG (Amersham Life Science) for 2 h at 37℃ . After having been washed five times in PBS, cells were mounted and analyzed by epifluorescence microscopy.
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RESULTS
1IN-sFv functional assay IN sFv-transduced SupT1 cells, CAT-,anti-Rev sFv-and anti-HBV core protein sFv-transduced SupT1 cells, and parental nontransduced SupT1 cells were infected with HIV-1 NL4-3 of the same MOI at 0.04～ 0.06. Syncytia and cell death and high levels of HIV-1 p24 antigen in the supernatants of nontransduced and CAT,anti-HBV core sFv transduced cells were observed at early time points after infection(10 d- 12 d), while there were only very low levels of HIV-1 p24 antigen in the supernatant of SupT1 cells transduced with IN sFv and few syncytia at the 30th- 34th day after infection(Figs 1,2,3).
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Fig 1 P24 level in the supernatant of NL4-3 challenged
SupT1 cells
Fig2 P24 level in the supernatant of NL4-3 challenged PBMCs
Fig 3 Results of NL4-3 virus challenged SupT1 cells(× 200)
A: Syncytia and dead cells of control SupT1 cells on 18th day;
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B: SupT1 cells transduced with IN- sFv show no syncytia and dead cells on 18th day.
PCR amplification of HIV-1 2-LTR-DNA HIV-1 2-LTR-DNA PCR amplification of 4,6, 8,16 h infected nontransduced SupT1 cells and transduced with IN-sFv and IN-sFv-nls cells showed that 2-LTR-DNA were detectable 8- 10 h earlier in the SupT1 expressioning IN-sFv and IN-sFv-nls than in the nontransduced SupT1 cells(Fig4). HIV-1 gag gene PCR amplification demonstrated that the virus quantity among the wells for cell culture was the same.
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Fig 4 Results of Southern blot
A： 2-LTR-DNA Southern blot with AA55 probe;
B： gag gene Southern blot with SK19 probe.
(S: SupT1 cells; IN: IN-sFv transduced SupT1
cells; NL: IN-sFv-nls transduced SupT1 cells).
Immunofloresence staining The indirect immunofluorescence staining of intracellular expressed IN-sFv and IN-sFv-nls demonstrated that the anti-IN-sFv was mainly cytoplasmic staining and nucleoplasm exclusion while the anti-IN-sFv-nls was nuclear staining(Fig 5).
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Fig 5 Results of immunofluo-rescence staining
A:SupT1 cells transduced with IN-sFv show cytoplasm positive × 1000;
B:SupT1 cells transduced with IN-sFv-nls show nuclear positive × 200.
DISCUSSION
Our results demonstrated that intracellularly expressed anti-HIV-1 IN-sFv significantly inhibited the production of HIV-1 p24 protein both in SupT1 and PBMCs and the inhibition rate was approximately 95% - 98% compared with that control cells(Fig 1). There were no syncytia and cell death 30 days after virus infection either in SupT1 or in PBMCs. To determine whether the observed decreases in HIV-1 p24 antigen levels correlated with a specific intracellular inhibition of HIV-1 integrase activity or were merely secondary to other effects of intracellular expression of anti-IN sFv in T-lymphoid cells, levels of HIV-1 2-LTR-DNA were compared by semiquantitative PCR. The binding domain of the IN-sFv used in this experiment was the carboxy terminus of the HIV-1 integrase which had been shown to account for the nonspecific DNA affinity of the protein, and might be involved in target DNA interactions[3,4]. When a specific residue in the carboxy terminus of IN, Trp-235, was replaced by Ala, the ability of the HIV-1 provirus to replicate was abolished[5]. As shown in Fig. 4, the detectable levels of HIV-1 2-LTR-DNA molecules occurred 8- 10 hours earlier in cells expressing anti-IN-sFv or anti-IN-sFv-nls than in the control cells, which indicated that the virus cDNA accumulated and was ligated into a circled 2-LTR-DNA after it entered the infected cell nuclear due to the blocking of its integrase by intracellularly expressed anti-IN-sFv and it could not integrate into the infected cell genome any more[6,7}.
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The quantitative changes of 2-LTR-DNA at the same time point after infection was similar to those in the experiment results conducted with mutant integrase[8]. The difference between the two experiment results was that 2-LTR-DNA appeared at least 2 hours earlier in anti-IN-sFv-nls transduced cells in which the sFv was located in the nucleus, than in the cells transduced with anti-IN-sFv in which the sFv was located in the cytoplasmic compartment. The time difference suggests that HIV-1 IN may be initially sequestered by the cytoplasmic sFv ,delaying nuclear importation of the preintegration complex, where viral DNA ends are joined via nuclear ligase activity. Alternatively,IN-sFv-nls may facilitate its transport into the nuclear compartment by virtue of binding to the preintegration complex.
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The results of this experiment further demonstrate that intracellularly expressed anti-HIV-1 IN-sFv can significantly inhibit virus replication via blocking the virus integrase and that the function of sFv is related to its intracellular localization. The sFv also serves as valuable reagents with which to explore further the combined target gene therapy methods in the blocking of HIV-1 replication.
Author： ZhangHZ,male， associate professor， Ph.D,M.D. Tangdu Hospital, Xinsi Road, Xi'an 710038, Shaanxi Province, China. Tel.(029)3577591
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REFERENCE
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〔7〕 Farnet CM, Haseltine WA. Circularization of human immunodeficiency virus type 1 DNA in vitro〔J〕 . J Virol, 1991; 65(12): 6942- 6952.
〔8〕 Engelman A, Englund G, Orenstein JM,et al. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication〔J〕 . J Virol,1995; 69(5): 2729- 2736.
Received date:1999- 07- 13
revised date:1999- 09- 13, http://www.100md.com