The Changes Of MRNA Expression Profile In Host Cells Infected With ORFV And The Mechanism Of ORFV-induced Cell Autophagy

The Orf virus, an epitheliotropic DNA virus, is the prototype species of theParapoxvirus genus of the poxvirus family, and it primarily causes contagiousecthyma, also known as Orf, in goats and sheep. The disease not only causes hugeeconomic losses to the sheep industry but also, as a zoonotic disease, poses a threat tohuman health. Currently, severe and prevalence of Orf has been gradually increased.In addition, the spectrum of host infection also gradually expanding, the disease hasbeen described in wild ruminants besides small ruminants. In recent years, genomicstructure, immunological properties and applications of carrier of ORFV have beenadvanced. However, there are no effective antiviral treatments available for Orf.It is a very complex process of infection and pathogenicity of virus. And it couldcause some differences in the expression of genes of host susceptible cells and altertheir mediated signaling pathways, consequently, resulting in cell physiologydysfunction leading and leading to the occurrence of the disease.In view of this, webuilt the mRNA expression profiles of differentiate expressing genes of ovine fetalturbinate (OFTu) cells-infected with ORFV48h and96h to screen differentiallyexpressed genes. The results of analysis showed that1047genes were up-regulated and366genes down-regulated at48h post infection(48h-pi);1570genes were up-regulatedand982genes down-regulated at96h-pi. GO analysis showed that the molecular functionof differentially expressed genes involved in the catalytic activity, binding activity,regulation of enzyme activity, molecular transduction activity and protein bindingtranscription factor activity; Biological processes of differentially expressed genes mainlyinvolved in immune processes, biological regulation processes, metabolism, stressresponse and so on. Pathway analysis showed differentially expressed genes mainlyinvolved in autophagy, apoptosis, inflammation, and immunity-related pathways. In aword, the study of differentially expressed genes provides an experimental basis forORFV and host interaction studies and a theoretical basis for further study of the molecular mechanisms of pathogenesis of ORFV.In recent years, many studies have demonstrated that there is a direct effectrelationship between autophagy and viral infection. However, the relationship ofORFV and autophagy remains largely unknown. According to the results ofbioinformatic analysis of differentially expressed genes, we chose the autophagy asstudy object, which plays an important role in the physiology and pathology as well asinnate immunity and acquired immunity. Autophagy is a major intracellular pathwayfor degradation and recycling of cytoplasmic organelles, damaged proteins andlong-lived proteins and that plays an essential role in maintenance of homeostasis.Therefore, the transmission electron microscopy, indirect immunofluorescence andimmunoblotting were carried out to detect autophagic morphology, localization ofendogenous LC3and LC3conversion at ORFV-infected host cells; Our resultsdemonstrated that viral infection can induce autophagy of OFTu cells or Helacells-infected with ORFV by the observation of autophagy utilizing electronmicroscopy and indirect immunofluorescence, and the conversion of LC3ofquantitative analysis by immunoblotting. In addition, we measured autophagic flux bymonitoring SQSTM1/p62degradation to confirm that ORFV infection induced thecomplete autophagic process. The results showed that ORFV can induce autophagyand autophagic lysosome fusion and thus degrade the substrate in infected host cells(OFTu cells and Hela cells).After confirm that ORFV infection induced the complete autophagic process, theOFTu cells and Hela cells were treated with different autophagy inhibitors (earlyinhibitor3-MA and late inhibitors CQ) or inducers (Rapa or EBSS), and analyzedthem effect on progeny virus yield. The results showed that whether at the effectiveinduction or inhibition of autophagy case, the infection titers of ORFV did not changesignificantly. The host could activate autophagy to protect themselves when virusesattack, while ORFV may also encode proteins to evade or resist the host antiviralresponse.In order to clarify the mechanism of ORFV infection induced the host cellautophagy. In this study, total protein of the Hela cells-treated with ORFV orRapamycin was extracted, then western blot analysis were performed; The resultsshowed that the level of phosphorylation of mTOR of ORFV infected group andRapamycin treatment group reduced, while the expression of LC3-Ⅱ significantly increased. That is to say that mTOR signaling pathway played an important roleduring the process of ORFV induced cell autophagy.To further validate the inhibition of mTOR pathway is a critical step for ORFVinduced autophagy, we performed site-directed mutagenesis of Rheb gene that couldactivate mTOR activity in order to construct Rheb activation form: pCMV-myc-RhebQ64L vector. The pCMV-myc-Rheb Q64L plasmids were transfected cells, then\immunoblot analysis were performed; our results suggested that the phosphorylationlevel of mTOR significantly increased, while the expression of LC3Ⅱdecreased in thecells-transfected Rheb Q64L plasmids; the results indicated that Rheb Q64L restoredactivity of mTOR. In order to validate that ORFV could induce autophagy byinhibiting mTOR pathway，the expression of Erk1/2and TSC2which situated atupstream of mTOR was detected，and found the Erk1/2and TSC2were activated. Thecells were treated with U0126, the inhibitor of Erk1/2, prior to virus infection, thenthe levels of phosphorylation of mTOR, Erk1/2and TSC2, and the expression ofLC3-Ⅱwere detected by immunoblot analysised; the results showed that the level ofp-Erk1/2, p-TSC2and LC3-Ⅱ significantly suppressed, while the p-mTOR levelsignificantly increased. After that, we designed and synthesised three pairs of siTSC2,and the inhibition efficiency of siTSC2-1, siTSC2-2and siTSC2-3was73.4%,65.4%and93.1%, respectively. The cells were treated with siTSC2prior to virus infection,then the levels of phosphorylation of mTOR, TSC2and Erk1/2were detected byimmunoblot analysised; and found that expression of p-TSC2and LC3-Ⅱ reducted,while p-mTOR upregulated, whereas p-Erk1/2didn`t significantly change. Theseresults suggested ORFV might induce autophagy via Erk/TSC2/mTOR signalingpathway in host cells. The achieved findings will help better understanding themolecular pathogenesis of ORFV infections.