| Porcine epidemic diarrhea virus（PEDV）is an enveloped,single-stranded RNA virus of the coronavirus family,which can infect all ages of swine and cause severe economic losses in the swine industry.Though various studies have discovered many aspects of PEDV infection,still little is known about how PEDV traverses across the dense cytoplasm micro-environment to the place for replication.In order to answer this question,single virus tracking method was used to study the intracellular transport mechanism of PEDV.In order to visualize the intercellular transport of PEDV by using single virus tracking,we successfully realized the fluorescent labeling of PEDV and dynamically analyzed the role of microtubule and motor proteins（dynein and kinesin-1）in the intracellular transport of PEDV.1 Monochromatic fluorescent labeling of PEDV using quantum dots and lipophilic dye Di DIn order to realize the real-time tracking of PEDV,we established a method of labeling PEDV with quantum dots.First,the QD-N3 was obtained by the amination reaction of QD-N2 and NHS-N3eater.Then,QD-PEDV was obtained by the click chemical reaction of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine n-[dibenzocyclooctyl（polyacetylene glycol）-2000]（DSPE-PEG-DBCO）and QD-N3.By detecting the PEDV titer before and after QD label,we found that there was no significant difference in the virulence of PEDV before and after QD labeling,indicating that this labeling method had no significant effect on PEDV infection.Meanwhile,we use a colocalization assay by incubating the antibody of PEDV N protein in the wild-type PEDV or QD-PEDV infected Vero cells to detect the specific label of QD to PEDV.And we found that QD signal could only be detected in QD-PEDV infected cells,and 81.7%±3.5%of the QD signals from QD-PEDV infected cells was collocated with the immunofluorescence signals of PEDV N protein,while,no co-localization signals were found in wild-type viruses,indicating that QD can specifically label to PEDV.At the same time,we also successfully used the lipophilic fluorescent dye Di D to label PEDV.Through analyzing the characterization of the labeled viruses,it is found that the fluorescence intensity of most Di D is below 70 a.u.,and the virus particles are also relatively uniform.Through analyzing the virulence of PEDV before and after Di D labeling,it was found that Di D had no significant effect on PEDV infection.The above two methods laid an important foundation for the dynamic analysis of PEDV infection in the later stage.2 The intercellular transport of PEDV moving along microtubulesAnalogous to the spread of viruses within the host animal during pathogenesis from the entry site to the distance through the blood flow,lymphatic system and nervous system in the host body,there is also movement within infected cells.As cytoplasmic diffusion only operates within very small volums,active membrane traffic and cytosolic transport of viral genome-protein complexes are required,and it has been reported that microtubules can mediate the transport of various viruses.Therefore,we used QD labeled PEDV method combined with single virus tracking technology to monitor the intercellular transport of PEV in live Vero cells.Based on the analysis of the trajectories,velocities and MSD of 60 effective QD-PEDV from 20 cells,it is found that the intercellular transport of PEDV relied on microtubules in the host cell,and the transport is location-specific,that is,it shows that most PEDV had specific motion along the MTs:in the cell membrane region,PEDVs performed restricted motion mode with relatively stable speed;while in the cytoplasm and microtubule organizing center（MTOC）regions,the velocity of PEDV both followed the slow-fast-slow pattern,but in directional-directional-restricted diffusion motion and restricted-restricted-directional diffusion mode,respectively.Moreover,we also found that there were some viruses that experienced the return movement in the cell membrane and cytoplasm.These studies fully reflect the complex process of PEDV movement in host cells and propose important references for further study on the PEDV infection mechanism.3 The effect of the intercellular transport of PEDV driven by dynein and kinesin-1 along microtubule on PEDV membrane fusion and accumulation in the perinuclear regionIt is reported that microtubules and its two motor proteins can mediate the directional and bidirectional movement of a variety of viruses in cells.Combine with our previous research findings,PEDV can move in directional and return movement in cells.In order to reveal whether the two motor proteins are involved in PEDV infection,the confocal microscopy experiments,drug inhibitors and si RNA knockdown experiments were used.It was verified that microtubule,dynein and kinesin-1 not only could interact with PEDV,but also could influence the titer of PEDV.In order to verify that how microtubules,dynein and kinesin-1 are involved in PEDV infection,we analyzed the fusion and accumulation in the perinuclear region events of PEDV,respectively.First,we first studied the membrane fusion event of PEDV.It is reported that the fluorescent signal of Di D will be amplified when the virus labeled with lipophilic dye Di D undergoes a membrane fusion event.Therefore,the effects of microtubule,dynein and kinesin-1 on PEDV membrane fusion was carried out based on this characteristic of Di D and through drug inhibitors and si RNA knockdown experiments.Not only that,we also used the immunocolocalization method to repeatedly verify the PEDV membrane fusion event and the result is consistent with the conclusion of the Di D fluorescence statistical experiment,which further illustrates that microtubule,dynein and kinesin-1 affect PEDV membrane fusion.Second,the accumulation of PEDV in the perinuclear region through inhibition the function of microtubule and dynein as well as knockdown the expression of the kinesin-1 in the cells and then the cells were infected with Di D-PEDV was analyzed.Through statistical analysis,it was found that,compared with the cells treated with DMSO or control si RNA,PEDV in the microtubule and dynein inhibited and as well as kinesin-1 knockdown cells was scattered and distanced from the nuclei,suggesting that the inhibition of microtubules and dynein as well as the knockdown the expression of KIF5B significantly reduced the PEDV accumulation in the perinuclear region.While,it is also possible that the inhibition of microtubules and dynein as well as the knockdown of KIF5B may also decrease the attachment and internalization of PEDV,thus decreasing intracellular transport and further PEDV fusion and accumulation in the perinuclear region.To this problem,the influence of microtubules,dynein and kinesin-1 on PEDV attachment and internalization should be analyzed.To further verify whether microtubules,dynein and kinesin-1 affect the attachment and internalization of PEDVs,Western blot and real-time PCR were adopted.And the results found that microtubule,dynein and kinesin-1 did not influence PEDV attachment and internalization.In conclusion,it is found that microtubules,dynein and kinesin-1 are not related to PEDV attachment and internalization,thus proving that the reduction of PEDV fusion and accumulation in the perinuclear region with microtubule and dynein inhibition as well as KIF5B knockdown is mostly induced by the decrease of PEDV intracellular transport.4 Dynamic dissection of dynein and kinesin-1 cooperatively mediated intercellular transport of porcine epidemic diarrhea coronavirus along microtubuleCombined with our previous research findings,PEDV can move in directional and return movement in cells,and microtubule,dynein and kinesin-1 are involved in PEDV infection,additional,they are also related to PEDV fusion and accumulation in the perinuclear region but not to PEDV attachment and internalization.In order to reveal whether microtubule,dynein and kinesin-1 are also involved in PEDV intercellular transport,we combine the single virus tracking method to real-time reveal the function of microtubule,dynein and kinesin-1 in PEDV intercellular transport.The cells transfected EGFP-microtubule and m KO2-dynein or EGFP-microtubule and m KO2-KIF5B were infected with Di D-PEDV and monitor the intercellular transport by single virus tracking.Through the statistical analysis,it is found that PEDV can co-localize with microtubule,dynein and kinesin-1 in real-time during intercellular transport.Not only that,PEDV can perform five different movement modes along the microtubule driven by dynein or kinesin-1:（1）unidirectional movement towards microtubule plus ends,（2）unidirectional movement towards microtubule minus ends,（3）bidirectional movement along the same microtubule,（4）bidirectional movement along different microtubules and（5）motionless state.Among these types,the functions of dynein and kinesin-1 in PEDV intercellular transport were further analyzed by single-virus tracking and found that dynein and kinesin-1 mainly transport PEDV to the minus and plus ends of the microtubules,respectively;meanwhile,they also can transport PEDV to the opposite ends of the microtubules different from their conventional transport directions and also coordinate the bidirectional movement of PEDV along the same or different microtubules through their cooperation. |
