| Virus is an acellular life composed of nucleic acid and capsid protein,which leech on the material and energy of host cells to finish its life activities.FMDV is the causative agent of foot-and-mouth disease,one of the serious animal diseases,it mainly recognizes three types of cell surface receptor molecules and internalize via clathrin or caveolin dependent endocytosis,then virus capsid disassembly in acid endosomes,genomic RNA is released to complete the virus replication activities.FMDV infection trigger and regulate cell autophagy pathway,the involvement and timing of viral proteins in autophagy and ATGs in the process of FMDV infection have attracted much attention.This thesis is built around the life cycle of FMDV to elucidate the molecular mechanism and biological significance of the dynamic changes of ATG16L1 at different stages of virus replication.At first,compared the culture characteristics of three FMDV classic vaccine(O/HN/CHA/93)strains,we observed that the three FMDVs have differences in receptor utilization via amino acid substitutions of capsid proteins,rHN compared with the tissue virus(MF6)and cell adaptation virus(BF9),it showed a high affinity for heparin(HP)on BHK-21 cells,although it could not use HS receptor to infect in CHO-K1 cells,and the three strains had only three amino acid differences in the structural protein VP1—4.And we use reverse genetics technology with O/HN/CHA/93 vaccine strain(rHN)full-length infectious cDNA(POFs)as skeleton plasmid attempt to prepare site-directed mutants which use only one type of receptor,to provide materials for discriminate virus adsorption,invasion and the detailed pathway of cell-entry.Unfortunately,the tripeptide motif RGD-KGE/RGG(integrin?JMJD6)in the G—H loop of VP1 prevent the survival of virus.Moreover,It was found that E1083K is a critical amino acid for the MF6?BF9 to expand the rang of cell tropism and the rHN obtain HP+property,and the L2080 co-evolution with E1083K in adapted to in vitro culture.K1083R could compensate the deleterious effects of L2080M and D1138G of rHN for the HS-property to infected CHO cell lines.The results of JMJD6 blocking assay and IFA implied that JMJD6 may not a receptor for FMDV.The HP+site-directed mutants co-locate with caveoline-1 on the surface of BHK-21 cells suggested that the structure of HS molecules on BHK-21 cells and CHO-K1 cells may different.Subsequently,we choose two different HP sensitivities cell adapt FMDVs to study the virus internalization pathway in BHK-21 cells.The results showed that O/HN/CHA/93(HP+)and O/Fujian/CHA/5/99(HP-)appeared in the early endosome(EE)after entry cells mediated by caveolin and clathrin respectively.Neither of the two FMDVs capsid proteins appeared in the late endosome(LE)and recycling endosome(RE),but co-located with trans-Golgi(TGN).Thus,transmission electron microscopy(TEM)observed compared with mock-infected cells,both double-or single-membrane vesicles structures were significantly increased after infected with O/Fujian/Cha/5/99 in BHK-21 cells.At the same time,IFA showed that punctuate LC3 significantly accumulated in FMDV infected cells,and the conversion from endogenous LC3-? to LC3-? increased with the production of FMDV structural proteins(SPs).HESS treatment for deprived of nutrition to induce autophagy was benefit for FMDV replication,but mTOR inhibitor rapamycin treatment to induce autophagy had no significant effect on FMDV protein synthesis.BHK-21 cells were treatment by autophagy inhibitors 3-MA,chloroquine and bafilomycin A1 were significantly reduced the FMDV replication.FMDV infection affects autophagy,and autophagy plays a favorable role in FMDV infection.Confocal microscopy observation showed the FMDV non-structural proteins(NSPs)2BC,3A and 3C co-locate with LC3,suggesting that these NSPs involved in the regulation of autophagy in FMDV infected BHK-21 cells.Finally,we notice that clathrin mediated internalization of ATG16L1-containing vesicles trafficking pathway and FMDV endosomal transport pathway are "parallel",the potential function of ATG16L1 in the early and late stages of FMDV infection and the effect of ATG16L1-FMDV interaction were studied.The results show that ATG16L1 knockdown was benefit for FMDV replication,and overexpression ATG16L1 decreased the FMDV replication.ATG16L1 was slightly up-regulated in the early stage of FMDV infected in BHK-21 cells,but was significantly down regulated in the late stage.When FMDV infected in BHK-21 cells,ATG16L1 endosomal position changed and sorted into EE to be parallel to the FMDV transportation pathway.Compared with the ATG16L1-deficient BHKATG6L1-/-cell line,we found that ATG16L1 increased the internalization of FMDV but have no effect on the adsorption in BHK-21 cells,it via vesicles transport to assist the FMDV uncoating and thus accelerate the process of infection.In the late stage of FMDV infection,ATG16L1 was degraded by 2BC through the caspase pathway which located in TGN.Rab33B recruited ATG16L1 to Golgi membrane trafficking through a direct interaction with ATG16L1,which affect the proliferation of FMDV.BHK-21 cells transfected with siRab33B or Rab33B dominant mutants increased FMDV replication,and pCMV-Rab33B decreased FMDV replication.In conclusion,FMDV induced or prevented the break of virions by ATG16L1 on different stage of infection,which is one of the molecular mechanisms that virus and host cell regulate each other to achieve progeny virus proliferation.This thesis lays a foundation for further exploring the molecular basis of adaptive co-evolution of FMDV in vitro and the causes of autophagy and non-autophagy functions of ATGs in the life cycle of FMDV.It enrich our cognition of the pathogen "hijacking" host(cell)factors to antagonize the body's"defense system"... |
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