成视网膜细胞瘤结合蛋白4在HIV-1潜伏细胞中的作用研究  

Role of retinoblastoma protein-associated protein 48 （RBBP4） in HIV-1 latency

作　　者：王娟[1] 杨劲[2] 吴南屏[3] Wang Juan Yang Jin Wu Nanping(Department of Clinical Laboratory, Tongde Hospital of Zhejiang Province, Hangzhou 310012, China The Affiliated Hospital of Hangzhou Normal University, Hangzhou 311121, China State Key Laboratory for Diagnosis and Treatment cf Infectious Diseases, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China)

机构地区：[1]浙江省立同德医院检验科,杭州310012 [2]杭州师范大学附属医院,311121 [3]浙江大学医学院附属第一医院传染病诊治国家重点实验室,杭州310003

出　　处：《中华微生物学和免疫学杂志》2017年第3期188-193,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金面上项目（81273313）;浙江省医药卫生科技计划项目（2017KY275）;浙江省科技厅科技条件建设项目（2014F10014）

摘　　要：目的探讨成视网膜细胞瘤结合蛋白4（retinoblastoma protein associated proteins 48, RBBP4）分子在HIV-1潜伏中的作用及其机制。方法25 ng/ml的肿瘤坏死因子（tumor necrosis factor alpha, TNF-α）和10 ng/ml的IL-2刺激HIV-1潜伏细胞CEM-Bru后，利用染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）分别研究细胞刺激前后RBBP4以及与RBBP4关联的组蛋白脱乙酰化酶1和2（histone deacetylase, HDAC1/2）在前病毒启动子长末端重复序列（long terminal repeat, LTR）上的结合变化。电转法对潜伏细胞进行RBBP4部分干扰后，利用ChIP检测HDAC1/2以及组蛋白H3乙酰化水平和RNA聚合酶Ⅱ （RNA polymerase Ⅱ，RNA PolⅡ） 在LTR的结合变化，RT-PCR检测HIV-1起始转录本和延长转录本的变化。结果在HIV-1潜伏细胞中RBBP4和HDAC1/2在LTR上结合与激活状态相比显著增加；将RBBP4部分干扰后，RBBP4在LTR上的结合明显减少，导致HDAC1和HDAC2在LTR上的结合也减少，并伴随着组蛋白3 （histone 3, H3）乙酰化水平的增加和RNA PolⅡ在LTR上结合的增加。此外，RBBP4干扰后HIV-1起始转录本明显增加。结论RBBP4能促进HIV-1潜伏，这种作用可能与组蛋白脱乙酰化酶HDAC1和HDAC2有关。Objective To investigate the role and mechanism of retinoblastoma protein-associated proteins 48 （RBBP4） in HIV-1 latency. Methods CEM-Bru cells latently infected with HIV-1 were stimu- lated with 25 ng/ml of tumor necrosis factor alpha （TNF-oL） in combination with 10 ng/ml of interleukin-2 （IL-2）. Chromatin immunoprecipitation （CHIP） was performed to detect the changes in RBBP4 and in his- lone deacetylases 1 and 2 （HDAC1/2） binding to long terminal repeat （LTR）. Binding activities of HDAC1/2 and RNA polymerase Ⅱ （RNA Pol Ⅱ） to LTR and acetylated histone H3 at LTR region were detected by ChIP after partially interfering the expression of RBBP4 in CEM-Bru cells with electroporation. Ini- tiating and elongated transcripts were measured by RT-PCR. Results The binding activities of RBBP4 and HDACI/2 to LTR in HIV-1 latently infected cells were enhanced significantly as compared with those in TNF-α and IL-2 co-stimulated cells. Fewer RBBP4 and HDAC1/2 bound to LTR following the interference of RBBP4 expression, which was accompanied with enhanced histone acetylation and strengthened binding activity of RNA Pol Ⅱ to LTR. Moreover, more initiating transcripts were detected in HIV-1 latently infected cells after the RBBP4 expression was interfered by electroporation. Conclusion RBBP4 contributes to the maintenance of HIV-1 latency, in which HDAC1 and HDAC2 might be involved.

关 键 词：RBBP4 HDACl HDAC2 HIV-1 潜伏 

分 类 号：R446.6[医药卫生—诊断学] R512.91[医药卫生—临床医学]