接头蛋白NHERF1在HIV-1病毒复制中的作用

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family.Given the nature of GPCRs and their propensity to form oligomers, one can consider ligand-based therapies as unselective in terms of the oligomeric composition of complexes.For example, a ligand targeting a CCR5 homomer could likely induce signal transduction on a heteromeric CCR5-CXCR4. Other avenues could therefore be explored. We identified a receptor adaptor interacting specifically with one receptor complex but not others. NHERF1, an adaptor known for its role in desensitization, internalization, and regulation of the ERK signalingcascade for several GPCRs, interacts via its PDZ2 domain with the CCR5 homodimer but not with the CXCR4-CCR5 heterodimer or CXCR4 homodimer. To further characterize this interaction, we also show that NHERF1 increases the CCR5 recruitment of arrestin2 following stimulation. NHERF1 is also involved in CCR5 internalization, as we demonstrate that coexpression of constructs bearing the PDZ2 domain can block CCR5 internalization. We also show that NHERF1 potentiates RANTES (regulated on activation normal T cell expressed and secreted)-induced ERK-1/2 phosphorylation via CCR5 activation and that this activation requires NHERF1 but not arrestin2. Taken together, our results suggest that oligomeric receptor complexes can associate specifically with partners and that inthis case NHERF1 could represent an interesting new target for the regulation of CCR5 internalization and potentially HIV infection.Given the importance of CCR5 internalization during HIV-1 infection, we evaluated NHERF1's contribution in HIV-1 infection. We challenged human osteosarcoma cells coexpressing CD4 and CCR5 cells expressing either NHERF1 fragment domains or WT NHERF1 with an HIV-1 strain to examine the effects of NHERF1 on HIV-1 entry and replication. WT NHERF1 potentiates HIV-1 envelope gp120-induced CCR5 internalization,and promotes the replication of HIV-1. In order to better understand how NHERF1 affects signal transduction, different domains of NHERF1 were overexpressed in cells to analyze their effect on the different signaling pathways. Here, we show that NHERF1 can associate with CCR5, and promote activation of the gp120-induced MAPK/ERK, focal adhesion kinase and RhoA (Ras homolog gene family member A) signaling pathways. NHERF1 overexpression also increases HIV-1 host cell migration triggered by gp120 via FAK.However,the function and involved mechanism of normal expression level ofendogenous NHERF1 remains unclear.In this proposal, we will investigate the effects of endougenous NHERF1 on HIV-1 replication level, the host cell signaling pathways and related cytoskeleton. The project is of great importance in revealing the roles of NHERF1 in HIV-1 replication, as well as the discovery and development anti-HIV drugs based on viral internalization.

NHERF1是细胞内一个重要的多功能接头蛋白。我们前期的研究证明，NHERF1可以结合HIV-1的辅助受体CCR5的同源二聚体，并上调CCR5嗜性HIV-1的包膜糖蛋白gp120特异性诱导的受体内化和信号通路的激活，以及HIV-1病毒的产生。但是，宿主细胞中内源性NHERF1对HIV-1感染复制的影响尚不完全清楚。本项目拟在前期的研究基础上，进一步研究内源性NHERF1在敲减水平下，HIV-1感染宿主细胞后病毒进入细胞的情况，以及在胞内的复制水平和病毒的产生；病毒感染后相关的宿主细胞信号通路的激活情况以及宿主细胞骨架的重排变化，从而阐明NHERF1在HIV-1感染和复制过程的调控机制。通过本项目，将明确NHERF1分子对HIV-1感染复制的作用和机制，为可能开发基于NHERF1分子的小分子抑制剂等新型抗病毒药物奠定基础。

趋化因子受体CCR5和CXCR4是人免疫缺陷病毒（HIV-1）进入宿主细胞所必需的两种主要共受体。NHERF1是一种细胞内重要的多功能接头蛋白。我们前期研究发现过表达NHERF1可以结合HIV-1共受体CCR5的同源二聚体，并上调CCR5嗜性HIV-1包膜糖蛋白gp120特异性诱导的受体内化和信号通路激活，以及HIV-1病毒产生。但是，宿主细胞中内源性NHERF1对HIV-1感染复制的影响尚不完全清楚。本项目中，我们通过shRNA慢病毒表达载体构建了稳定敲减内源性NHERF1表达的HIV-1易感细胞系，进一步研究了内源性NHERF1在敲减水平下，HIV-1感染宿主细胞后CCR5受体和病毒进入细胞的情况，以及病毒在胞内的复制水平和病毒颗粒的产生情况；病毒感染后相关的宿主细胞信号通路的激活情况以及宿主细胞骨架的重排变化，从而阐明NHERF1在HIV-1感染和复制过程的调控机制。本课题圆满完成项目任务书的研究内容，主要结果和结论如下：（1）、我们鉴定出2个NHERF1特异性shRNA，Lenti-shRNA可以稳定降低宿主细胞内源性NHERF1表达水平，阻断共受体CCR5的内化作用，从而提高CCR5在细胞质膜表达水平；同时，NHERF1特异性shRNA可以限制CCR5嗜性HIV-1病毒的复制；研究还表明，NHERF1不识别HIV-1的衣壳蛋白，因此其本身没有限制或促进病毒复制活性。（2）、NHERF1特异性shRNA显著下调了HIV-1诱导的MAPK/Erk和cofilin磷酸化，以及对FAK磷酸化和RhoA信号通路活化有一定程度的影响，提示NHERF1通过调控胞内逆转录和运输来介导对病毒复制活性的影响。进一步对病毒复制产物的q-PCR实验表明，NHERF1在HIV-1逆转录后、PIC复合物转核和整合之前的阶段调控病毒的复制。（3）、通过激光共聚焦显微镜研究显示，在NHERF1 shRNA稳定表达的细胞内，HIV-1病毒感染后细胞内骨架蛋白F-actin发生重排减弱，证明NHERF1参与调控胞内的细胞骨架蛋白的重排来调控病毒的复制，特别是病毒PIC复合物的胞内运输。综上，本研究项目确定了NHERF1在HIV-1病毒复制中的作用，揭示了该分子在HIV-1感染复制过程中的可能调控机制，为基于NHERF1等共受体调控分子的新型抗病毒药物的开发奠定了基础。