趋化因子RANTES和SDF-1细胞内共表达抑制HIV-1感染的研究

Inhibition of HIV-1 infection by phenotypic knockout of HIV-1 chemo kine coreceptor CXCR4 and CCR5

目的 观察HIV 1辅受体CCR5和CXCR4的配体在细胞内共表达抑制HIV 1感染的作用。方法 应用磷酸钙沉淀法共转染HIV 1辅受体及其配体的质粒 ,制成辅受体表型剔除的靶细胞 ,与转染HIV 1膜蛋白质粒的细胞混合 ,观察合胞体形成并记数 ;脂质体介导法将含有报告基因CAT而缺失HIV包膜蛋白的质粒与HIV包膜蛋白质粒共转染 2 93细胞 ,包装成具有一次感染活性的假病毒 ,感染转化pCMV R K S K、pCMV R K、pCMV S K或pCMV的PM 1细胞 ,采用同位素薄层层析分析法检测CAT活性。结果 pCMV R K S K转染可以显著抑制M及T嗜性HIV膜蛋白诱导的合胞体形成 ;CAT检测发现与pCMV转染组相比 ,当两种嗜性重组病毒感染pCMV R K S K转染组PM 1细胞时 ,仅检测到背景水平的CAT活性。结论 HIV 1辅受体CCR5 CXCR4表型剔除可以明显抑制M和T嗜性HIV

Objective To observe the inhibition of HIV-1 in fection by coinactivating genetically both CCR5 and CXCR4 in vitro. Methods HeLa-T4 + cells cotransfected with HIV-1 coreceptors p lasmid DNA ( pCMV -CCR5 or pCMV-CXCR4) and chemokine expression plasmid DNA (pCMV-R-K-S-K, pCMV-R-K or pCMV-S-K) using a calcium phosphate system we re cocultured with HeLa cells transfected with HIV-1 envelope protein expressin g plasmids. Syncytia in each well was counted after crystal violet staining, and the percentages of inhibition were calculated. The cells were cotransfected wit h pHIV-ΔenvCAT and pHIV-env(pADA, pYU2 or pIIIB) by Lipofectin. The recombina nt viruses were used to infect target cells, and 60 h later, the target cells we re lysed and used for determination of CAT activity. Results By cotransfection with pCMV-R-K-S-K, M and T tropic envelope-mediated s yncytium formation was significantly inhibited. By infection with the recombinan t M or T-tropic viruses, high levels of CAT activity were detected in PM-contr ol, but only background levels of CAT activity were detected in PM-1 cells tran sfected with pCMV-R-K-S-K. Conclusion Coexpression of CC-and CXC-intracellular chemokines blocked both M- and T-tropic HIV-1 ent ry into the transduced cells.