The host cell ubiquitin ligase protein CHIP is a potent suppressor of HIV-1 replication
The host cell ubiquitin ligase protein CHIP is a potent suppressor of HIV-1 replication
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DOI：
10.1074/jbc.ra118.007257
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发表时间：
2019-05-03
影响因子：
4.8
通讯作者：
Banerjea, Akhil C.
中科院分区：
文献类型：
--
作者：
Ali, Amjad;Farooqui, Sabihur Rahman;Banerjea, Akhil C.
人类免疫缺陷病毒-1（HIV-1）达特在宿主细胞中通过蛋白酶体和溶酶体途径降解，但与来自这些途径的达特结合的特异性分子尚不清楚。由于E3泛素连接酶是泛素依赖性蛋白酶体降解蛋白质的底物特异性的主要决定因素，我们首先试图鉴定与达特降解相关的E3连接酶。基于达特蛋白的内在无序性，我们将研究的重点放在了宿主细胞E3泛素连接酶CHIP（HSP 70-binding protein的C末端）上。共转染达特与CHIP表达质粒的达特蛋白的水平以剂量依赖性方式降低，而不影响相应的mRNA水平。此外，在CHIP存在下，通过放线菌酮（CHX）追踪测定测量的达特蛋白降解速率增加。缺乏负责蛋白泛素化的U盒结构域（CHIPU盒）的CHIP突变体不能降解达特蛋白。此外，CHIP通过WT以及在位置48处仅具有单个赖氨酸残基的Lys-48-泛素促进达特的泛素化。在HIV-1报告TZM-bl细胞中的CHIP转染导致Tat依赖性HIV-1长末端重复序列（LTR）启动子反式激活以及HIV-1病毒体产生减少。使用CRISPR-Cas9在HEK-293 T细胞中敲低CHIP导致更高的病毒体产生和增强的Tat介导的HIV-1 LTR启动子反式激活，沿着达特蛋白的稳定化。总之，这些结果表明宿主细胞E3泛素连接酶蛋白CHIP通过其调节蛋白达特的泛素依赖性降解在调节HIV-1复制中的新作用。
Human immunodeficiency virus-1 (HIV-1) Tat is degraded in the host cell both by proteasomal and lysosomal pathways, but the specific molecules that engage with Tat from these pathways are not known. Because E3 ubiquitin ligases are the primary determinants of substrate specificity within the ubiquitin-dependent proteasomal degradation of proteins, we first sought to identify the E3 ligase associated with Tat degradation. Based on the intrinsic disordered nature of Tat protein, we focused our attention on host cell E3 ubiquitin ligase CHIP (C terminus of HSP70-binding protein). Co-transfection of Tat with a CHIP-expressing plasmid decreased the levels of Tat protein in a dose-dependent manner, without affecting the corresponding mRNA levels. Additionally, the rate of Tat protein degradation as measured by cycloheximide (CHX) chase assay was increased in the presence of CHIP. A CHIP mutant lacking the U-box domain, which is responsible for protein ubiquitination (CHIPU-box), was unable to degrade Tat protein. Furthermore, CHIP promoted ubiquitination of Tat by both WT as well as Lys-48-ubiquitin, which has only a single lysine residue at position 48. CHIP transfection in HIV-1 reporter TZM-bl cells resulted in decreased Tat-dependent HIV-1 long-terminal repeat (LTR) promoter transactivation as well as HIV-1 virion production. CHIP knockdown in HEK-293T cells using CRISPR-Cas9 led to higher virion production and enhanced Tat-mediated HIV-1 LTR promoter transactivation, along with stabilization of Tat protein. Together, these results suggest a novel role of host cell E3 ubiquitin ligase protein CHIP in regulating HIV-1 replication through ubiquitin-dependent degradation of its regulatory protein Tat.