Early Entry Mechanism Of Porcine Epidemic Diarrhea Virus Infection Unraveled By Single-Virus Tracking

Porcine epidemic diarrhea virus(PEDV),a member of the genus Alphacoronavirus in the family Coronaviridae,causes acute diarrhea and/or vomiting,dehydration,and high mortality in neonatal piglets.Single-virus tracking is an important tool to visualize the dynamic of biological events.It can monitor the dynamic of a single virus infecting the living cells in real-time,and analyze the dynamic characteristics,such as viral speed,trajectory and movement mode during the virus infection.Single-virus tracking has become an effective method for studying the dynamic mechanism of virus infection.The early entry of envelope virus is one of the most important steps in viral infection.In this study,we used single-virus tracking to dissect the key events of PEDV early entry stage,such as endocytic pathways and membrane fusion of PEDV at single virus level.This will further reveal the mechanism of PEDV’s early cell entry mechanism in time and space.In addition,the combination of overexpression mutant plasmids,electron microscopy and immunoelectron microscopy,we further verified the endocytic pathways and endosomes that PEDV relies on in the early stages of viral entry.These results lay an important theoretical foundation for the prevention and control of PEDV infection.In this study,we carried out experiments in the following four aspects:1.Mechanism of PEDV entry into the cells via clathrin-mediated endocytosis(CME)CME is the main pathway for substances to enter cells,and it is also one of the most common endocytic pathways for viral infection.Studies have shown that PEDV can hijack CME to enter cells,however up to now,the spatiotemporal dynamic mechanism of PEDV enter into cells via CME is still unclear.Basing on single-virus tracking,we dissected the spatiotemporal dynamic mechanism of PEDV entry via CME at single virus level.After PEDV adheres to the cells,it can recruit de-novo formation of CCPs at viral binding sites,and after CCPs disappears,PEDV enters the cells to complete the successful infection.Meanwhile,by overexpressing the mutant plasmid to disrupt the CME,and the entry of PEDV is also significantly reduced.In PEDV infected cells,TEM was also used to analyze the viral infection,and PEDV can induce the formation of CCPs at viral binding sites,this also indicating that PEDV can enter the cells via CME.In addition,basing on single-virus tracking,we found that PEDV can enter the cells without the formation of CCPs.This results prompt that PEDV may also have other endocytic pathways to enter the cells.Collectively,this study revealed the temporal and spatial dynamic mechanism of PEDV via CME for the first time,and it was found that CME was not the only endocytic pathway for PEDV to enter cells,which enhanced our understanding of the cell entry mechanism of PEDV.2.Mechanism of PEDV entry into the cells via caveolae-mediated endocytosis(Cav ME)Cav ME is one of the common endocytic pathways that cargos enter the cells via clathrin-independent endocytic pathways.Up to now,whether PEDV can enter the cells via Cav ME is still controversial.Single-virus tracking can intuitively and truly reveal the entire process of viral entry at a single virus level.In this study,using single-virus tracking,we tracked the infection of Di D-PEDV in cells transfected with Cav1-m KO2 plasmids in real-time at a single virus level.We found that after PEDV adheres to the cells,the virus can recruit the de novo formation of caveolae,and then Cav1-m KO2 labeled caveolae will enter the cells accompanied with PEDV entry into the cells,confirming that PEDV can enter into cells via Cav ME.In addition,overexpressing Cav1 DN and Cav3 DN plasmids to disrupt Cav ME,we found that the entry of PEDV was also significantly reduced,this results also prove that PEDV can enter cells via Cav ME.Using TEM,the ultrastructure of the cell membrane in PEDV infected cells was analyzed,it was found that PEDV could induce the formation of caveolae at the cell membrane during the process of PEDV penetrating the cell membrane barrier.In conclusion,this study confirms that PEDV can rely on Cav ME to enter cells,and resolves the previous controversy about whether PEDV can rely on Cav ME to enter cells.3.Mechanism of PEDV entry into the cells via clathrin and caveolae cooperatively mediated endocytosis(C~3ME)To further reveal the endocytic pathways that PEDV enters the cells,in cells co-expressed with Cla-EGFP and Cav1-m KO2 plasmids,we tracked the dynamic entry of Di D-PEDV in real-time.Firstly,basing on the three-color live cell imaging with confocal microscopy,we found that CCP and caveolae can co-mediate PEDV into cells.Secondly,basing on the three-color live cell imaging with total internal reflection fluorescence microscopy(TIRFM),it was further confirmed that the co-localization of CCP,caveolae and PEDV occurring at the cell membrane during PEDV entry.Moreover,our results also prove that PEDV can enter the cells via CME alone or Cav ME alone.Subsequently,the PEDV-infected cells were analyzed by double immunoelectron microscopy labeled with clathrin and caveolin1,and it was found that PEDV can induce the formation of CCP,caveolae,and clathrin-caveolin1 double positive invagination,indicating that PEDV can enter cells via CME,Cav ME and C~3ME,respectively.In addition,there are 20%of PEDV existing abortive entry via CME and Cav ME that PEDV still stays at the cell membrane after the disappear of CCP or caveoale.we didn’t find the abortive entry of PEDV via C~3ME,indicating that C~3ME of PEDV may be a more efficient pathway.In short,PEDV enters the cells via C~3ME,and the abortive entry of PEDV via CME and Cav ME,which expands the theoretical basis of PEDV’s cell entry mechanism.4.Mechanism of PEDV entry via early and late endosomesEndosome is an important sorting place for viruses to enter cells through endocytosis.To explore the intracellular transport of PEDV in endosomes after entering into the cells,we directly tracked the infection of PEDV in cells co-expressing Cla-EGFP and Rab5-m Cherry plasmids,or in cells co-expressing Cav1-m KO2 and Rab5-EGFP plasmids.We found that once PEDV completed its internalization,viruses were quickly transported to the early endosomes.In cells co-expressing Rab5-EGFP and Rab7-m Cherry plasmids,we found that PEDV can be transported from the early endosomes to the late endosomes.In addition,overexpression of Rab5 DN or Rab7DN plasmid in cells to disrupt the formation of early endosomes or late endosomes,it was found that the internalization of PEDV was also significantly reduced.Also,our TEM results further prove that the early infection of PEDV is dependent on the early endosomes and the late endosomes.To further explore the main sites of PEDV membrane fusion occurring in endosomes,in cells co-expressing Rab5-EGFP and Rab7-m Cherry plasmids,we directly tracked PEDV membrane fusion in endosomes in real-time.We found that most of viral fusion occurring in late endosomes.Moreover,after PEDV co-localizes with the endosomes,viral fusion can occur within 6.8 minutes.In general,our study confirmed that early endosomes and late endosomes are important for PEDV infection,and we revealed the temporal and spatial dynamic mechanism of PEDV membrane fusion,which will improve our understanding of PEDV’s endosome transport and membrane fusion.