Inhibition of Tat-mediated transactivation and HIV-1 replication by human anti-hCyclinT1 intrabodies
Inhibition of Tat-mediated transactivation and HIV-1 replication by human anti-hCyclinT1 intrabodies
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DOI：
10.1074/jbc.m208297200
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发表时间：
2003-01-17
影响因子：
4.8
通讯作者：
Marasco, WA
中科院分区：
文献类型：
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作者：
Bai, JR;Sui, JH;Marasco, WA
人类免疫缺陷病毒1型（HIV-1）复制需要达特蛋白与正转录延伸因子（P-TEFb）复合物的人细胞周期蛋白T1（hCyclinT1）亚基相互作用，然后协同结合反式激活反应元件（TAR）RNA以反式激活HIV转录。本研究利用非免疫性人源性单链抗体（sFv）噬菌体展示文库，筛选出能破坏hCyclinT1-达特相互作用的抗hCyclinT1 sFv。将完全支持达特反式激活的hCyclinT1的N-末端272个残基（包括整个细胞周期蛋白结构域和Tat-TAR识别基序（TRM））用于淘选，并且在获得的五种独特的抗hCyclinT1 sFv中，三种结合细胞周期蛋白盒结构域，两种结合TRM。所有sFv都可以在瞬时转染的293T细胞和稳定的Jurkat和SupT1转染子中以高水平表达为胞内抗体，并且可以特异性地共免疫沉淀293T细胞中共表达的hCyclinT1，具有不同的功效，而不破坏hCyclinT1-Cdk9相互作用。此外，两个sFv克隆（3R6 - 1和2R6 - 21），映射到细胞周期蛋白盒结构域显着抑制Tat介导的反式激活在几个瞬时转染的细胞系，而不抑制基础转录或诱导凋亡。当在稳定的表达3R6 - 1的Jurkat T细胞上进行HIV-1攻击研究时，在低攻击剂量下获得了对病毒复制的几乎完全抑制，并且在SupT1细胞中在高感染剂量下实现了对HIV-1复制的74 - 88%抑制。这些结果提供了原则上的证据，抗hCyclinT1胞内抗体可以被设计成阻断HIV-1复制而不引起细胞毒性，因此，它们可能是用于HIV-1感染/AIDS的基于"细胞内免疫"的基因治疗策略的有用试剂。
Human immunodeficiency virus, type 1 (HIV-1) replication requires the interaction of Tat protein with the human cyclinT1 (hCyclinT1) subunit of the positive transcription elongation factor (P-TEFb) complex, which then cooperatively binds to transactivation response element (TAR) RNA to transactivate HIV transcription. In this report, a non-immune human single-chain antibody (sFv) phage display library was used to isolate anti-hCyclinT1 sFvs that could disrupt hCyclinT1-Tat interactions. The N-terminal 272 residues of hCyclinT1, including the entire cyclin domains and the Tat-TAR recognition motif (TRM), that fully support Tat transactivation was used for panning, and of the five unique anti-hCyclinT1 sFvs that were obtained, three bound to the cyclin box domains and two bound to TRM. All sFvs could be expressed as intrabodies at high levels in transiently transfected 293T and in stable Jurkat and SupT1 transfectants and could specifically co-immuno-precipitate co-expressed hCyclinT1 in 293T cells with varying efficacy without disrupting hCyclinT1-Cdk9 interactions. In addition, two sFv clones (3R6-1 and 2R6-21) that mapped to the cyclin box domains markedly inhibited Tat-mediated transactivation in several transiently transfected cell lines without inhibiting basal transcription or inducing apoptosis. When HIV-1 challenge studies were performed on stable 3R6-1-expressing Jurkat T cells, near complete inhibition of viral replication was obtained at a low challenge dose, and 74-88% inhibition to HIV-1 replication was achieved at a high infection dose in SupT1 cells. These results provide proof-in-principle that anti-hCyclinT1 intrabodies can be designed to block HIV-1 replication without causing cellular toxicity, and as a result, they may be useful agents for "intracellular immunization"-based gene therapy strategies for HIV-1 infection/AIDS.