手足口病（Hand, foot, and mouth disease,HFMD）是一种高度传染性疾病，多发于幼儿，其已成为全球性公共卫生安全问题，特别是在亚太地区尤为严重。手足口病的主要病原体是肠道病毒，尤以肠道病毒71型（Enterovirus 71, EV71）和柯萨奇病毒-A16（Coxsackievirus A 16, CVA16）最为常见。目前，人们对EV71和CVA16的致病机理并不清楚，也没有开发出特效药用来治疗因EV71或CVA16感染而引发的手足口病。

肠道病毒通常利用宿主细胞的转运或是代谢途径进行自身的复制，目前对于EV71/CVA16病毒如何利用宿主系统进行复制有待进一步阐明。为了系统性地筛选影响EV71感染的宿主因子，我们应用CRISPR-Cas9高通量筛选技术获得调控EV71感染的宿主候选因子，包括LAMTOR3。我们进而在HeLa和RD细胞中敲除LAMTOR3，发现LAMTOR3的缺失显著地抑制了由EV71或CVA16引起的细胞死亡。LAMTOR3属于Ragulator复合物中的一个亚基，Ragulator由LAMTOR1、LAMTOR2、LAMTOR3、LAMTOR4和LAMTOR5组成。我们发现敲除LAMTOR1或者沉默LAMTOR2或LAMTOR4的表达均能显著地抑制EV71引起的细胞死亡。进一步的研究发现，Ragulator的缺失不能抑制EV71与细胞的结合，也不能抑制EV71进入到细胞中，但Ragulator的缺失显著地抑制EV71病毒RNA的复制。Ragulator作为鸟苷酸交换因子可以活化Rag复合物，我们进一步发现RagB的缺失同样能抑制EV71病毒RNA的复制。这些结果表明Ragulator-Rag对于EV71病毒的复制是需要的。mTORC1复合物可以通过Ragulator-Rag复合物定位到溶酶体上，我们发现EV71感染能促进mTORC1活化，抑制mTORC1的活性降低了EV71病毒的滴度，表明Ragulator-Rag 促进mTORC1的活化有利于EV71病毒的复制。

为了进一步探究Ragulator-Rag复合物对EV71复制的调控，我们通过体外免疫共沉淀、邻位连接技术（PLA）以及免疫荧光技术等发现EV71的3D蛋白能够被Ragulator-Rag复合物招募到溶酶体表面。Ragulator-Rag复合物是通过LAMTOR1锚定于溶酶体，LAMTOR1 N末端的G2、C3和C4氨基酸是其锚定于溶酶体需要的。我们将LAMTOR1的G2、C3和C4全部突变为A, 构建LAMTOR1-3A突变体，会导致Ragulator复合物不能锚定到溶酶体上。在表达LAMTOR1-3A突变体的细胞中，EV71的3D蛋白不能定位到溶酶体上，且EV71病毒RNA的复制受到显著地抑制。同样地，Ragulator-Rag复合物对CVA16的复制也是必需的。

为了研发抗EV71病毒的新型药物，我们实验室在之前的研究中曾筛选出了抑制EV71复制的小分子化合物。我们发现其中一个化合物ZHSI-318不仅能够抑制EV71和CVA16病毒的复制，而且显著地抑制EV71和CVA16的3D蛋白锚定到溶酶体上。EV71感染引发的宿主细胞焦亡被认为是病毒致病的重要机制。我们发现ZHSI-318能够显著地抑制EV71病毒引发的细胞焦亡。而且，体内研究发现ZHSI-318能够有效抑制小鼠体内EV71的复制，表明ZHSI-318具有治疗EV71感染的潜力。因此，我们的研究表明ZHSI-318可以作为治疗由EV71或是CVA16引起的手足口病的潜在药物。

Hand, foot and mouth disease (HFMD) is a highly contagious disease among young children worldwide. It has become a global public health safety problem, especially in the Asia-Pacific region. The main pathogens of HFMD are enteroviruses, among which Enterovirus-A71 (Enterovirus 71, EV71) and Coxsackievirus-A16 (Coxsackievirus A 16, CVA16) are the most common viruses. At present, the pathogenic mechanism of EV71 and CVA16 is not fully understaood. There is no effective anti-enterovirus drug that has been developed to treat HFMD caused by EV71 or CVA16 infection.

Enteroviruses usually use host cell trafficking or metabolic pathways to facilitate viral replication. However, how EV71/CVA16 virus manipulates the host system for viral replication remains to be further elucidated. In order to systematically screen the host factors involved in EV71 infection, we performed a genome-wide CRISPR-Cas9 knockout screen and identified candidate host factors, including LAMTOR3. We then knocked out LAMTOR3 in both HeLa and RD cells and found that LAMTOR3 deficiency significantly inhibited cell death caused by the infection of EV71 or CVA16. LAMTOR3 is a subunit of the Ragulator complex, which consists of LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4, and LAMTOR5. Either deletion of LAMTOR1 or silenceof LAMTOR2 or LAMTOR4 significantly inhibited EV71-induced cell death. Furthermore, we found that the deletion of Ragulator did not inhibit the binding of EV71 to cells and the entry of EV71 into cells. Notably, the deletion of Ragulator significantly inhibited the replication of EV71 RNA. As a guanylate exchange factor, Ragulator can activate the Rags complex. We further found that the deletion of RagB also inhibited the replication of EV71 RNA. These results suggest that the Ragulator-Rag complex is required for EV71 replication. mTORC1 is localized on the surface of the lysosome near the cytoplasm through the Ragulator-Rag complex. We obsereved that EV71 infection induced mTORC1 activation. Inhibition of mTORC1 activity reduced the viral titer of EV71, indicating that the Ragulator-Rag complex promotes mTORC1 activation to facilitate EV71 replication.

To further explore the regulation of EV71 replication by the Ragulator-Rag complex, we found that the 3D protein of EV71 was recruited to the surface of lysosomes by Ragulator-Rag complexes using multiple technologies including co-immunoprecipitation, proximity ligation (PLA) and immunofluorescence. The Ragulator-Rag complex is anchored to the lysosome via LAMTOR1, which requires G2, C3 and C4 amino acids at the N-terminus of LAMTOR1. We generated a mutant form of LAMTOR1 in which G2, C3, and C4 were mutated to alanine (LAMTOR1-3A) that lost the ability to anchor to the lysosome surface. In the cells expressing LAMTOR1-3A mutant, the 3D protein of EV71 failed to localize to the lysosome surface, and the replication of EV71 viral RNA was significantly inhibited. Moreover, the Ragulator-Rag complex is required for CVA16 replication. Thus, our results demonstrate that the Ragulator-Rag-3D axis mediates the replication of EV71 and CVA16.

To develop new anti-EV71 drugs, a previous study in our laboratory had screened a library of small-molecule compounds and identified candidate compounds that affected EV71 replication. We found that one of the candidate compounds (ZHSI-318) not only inhibited the replication of EV71 and CVA16 viruses, but also significantly reduced the anchoring of 3D of EV71 and CVA16 to the lysosome surface. Pyroptosis of EV71-infected cells is considered as an important mechanism of viral pathogenesis. We found that ZHSI-318 significantly inhibited EV71 virus-induced pyroptosis. Moreover, in vivo experiments showed that ZHSI-318 effectively blocked the replication of EV71 in mice, indicating that ZHSI-318 has the potential to protect mice against EV71 infection. Therefore, our study indicates that ZHSI-318 has the potential for use in the development of anti-viral therapyfor the treatment of HFMD.