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。

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）

（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.

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.

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.

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.

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.

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.

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).

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,