Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90β siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90β resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90β and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. In vivo studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection.

尽管已有多项研究报道了参与肠道病毒71型（EV71）入侵与复制的相关因子，但介导这两个过程的确切分子机制仍未完全阐明。本研究证实，热休克蛋白90（HSP90）是介导EV71在人横纹肌肉瘤RD细胞中入侵与复制的关键因子。通过使用靶向HSP90β的小干扰RNA（siRNA）预处理宿主细胞、使用HSP90特异性抗体或格尔德霉素（geldanamycin, GA，一种HSP90特异性抑制剂）阻断HSP90功能，以及重组HSP90β处理，均可抑制病毒入侵及后续的病毒复制过程。重组HSP90β与EV71的免疫共沉淀实验，以及EV71与HSP90在细胞内的共定位分析，均证实HSP90可与EV71病毒颗粒发生直接物理结合。HSP90似乎通过抑制新合成的衣壳蛋白的蛋白酶体降解来介导EV71复制，但并未在转录水平上促进病毒基因的表达。通过GA对宿主细胞进行后置处理的实验可验证这一点：GA不会影响病毒转录本的表达，但会加速病毒衣壳蛋白的降解，并干扰组装成熟病毒颗粒的形成。本研究采用表达人清道夫受体B族2（SCARB2）的转基因小鼠，开展体内实验以评估HSP90抑制剂17-烯丙基氨基-17-去甲氧基格尔德霉素（17-AAG，格尔德霉素的类似物，其活性与GA相近但毒性更低）所提供的保护效果。实验结果显示，两次给药17-AAG可使表达人SCARB2的转基因小鼠抵御C2、C4及B4基因型EV71的感染攻击。本研究数据证实，HSP90在EV71感染过程中发挥重要作用。使用临床可用药物靶向HSP90，或可为EV71感染的治疗提供一种可行的治疗策略。