| 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. |
