The Transcriptional And Proteomic Differences Of Cells Infected With SPV And GPV And The Role Of K3L In Host Range Differences Of Capripox Virus

Capripox virus(CPV)is one of the most important animal poxviruses,which has host specificity.Under the natural condition,Goatpox virus(GPV)is able to infect both sheep and goats,while most of Sheeppox virus(SPV)strains infects only sheep,but the mechanism of this difference in infection remains unclear.In this study,the changes of transcriptome and proteome of Vero cells infected with either SPV or GPV from the perspective of host were systematically studied by high-throughput transcriptome sequencing and SILAC-MS/MS quantitative proteomics.Then,the effects of SPV infection on host PKR signaling pathway and the role of K3 L protein in regulating host antiviral pathway were analyzed by molecular biology experiments.Finally,the PKR of sheep and goat were analyzed by homologous modeling and molecular docking.The bioinformatics study of the factor eIF2?and K3 L protein of poxvirus SPV/GPV was carried out.The main findings are as follows:1.Affymetrix full transcript microarray was used to analyze the transcript expression profiles of Vero cells infected with SPV and GPV.In a total of 45763 molecules were detected,the transcriptome information of both SPV-infected cells and GPV-infected cells were compared and analyzed.Both GPV and SPV caused significant changes in cell transcriptome in the early stage of infection,but there was no significant difference between them.However,the signal of differential molecules in the virus-infected group was significantly different from that in the control group,and the differential molecules increased gradually with the increase of infection time.The differential molecules between GPV infection group and SPV infection group were also different,and changed significantly with the infection time in the late stage of infection.2.In this study,the proteomic differences of GPV infected host cells were quantitatively studied based on SILAC.Among 2146 proteins detected in the cytoproteome,110 proteins were up-regulated and 128 down-regulated by SPV infection;99 proteins were up-regulated and 77 were down-regulated by GPV infection,77 proteins were up-regulated after SPV infection and 70 down-regulated compared with GPV infection group.There were 27 up-regulated proteins in GPV infection and 31down-regulated proteins in SPV infection.Moreover,the up-and down-regulation multiple caused by GPV infection was higher than that caused by SPV infection.These results suggest that both GPV and SPV infection can cause significant changes in host cytoproteome levels.3.By using molecular biological methods,it was showed that SPV could activate PKR and inhibit the replication of SPV by inhibiting the total translation and stimulating the production of IFN-beta.CPV K3 L can interact directly with PKR and antagonize PKR.More importantly,the results of this study also showed for the first time that all CPVs K3 L homologues could inhibit PKR in sheep.GPV K3 L can strongly inhibit PKR in sheep and goats,and SPV K3 L only partially inhibits PKR in goats.These results further suggest that the degree of inhibition of PKR by CPV K3 L homologue may be related to the different host ranges of CPV in nature.4.By bioinformatics,the protein kinase PKR,the eukaryotic translation initiation factor eIF2? of sheep and goat and the K3 L protein of poxvirus SPV/GPV were systematically analysed.The bindingmode between them was determined and the potential key amino acids were identified and mutated by virtual mutation method.Through in-depth analysis of the interacting amino acids in the complexes,it is found that electrostatic interactions,especially hydrogen bonding interactions,play a major role in maintaining the stability of the complexes,whether in sheep or goat,or in PKR-eIF2? or PKR-K3 L complexes,and the interaction between the complexes is estimated in the CHARMM force field.It is found that the order of interaction energies is: GPV K3L-Goat PKR>GPV K3L-Sheep PKR >SPV K3 LSheep PKR>Goat eIF2?-Goat PKR>Sheep eIF2?-Sheep PKR.The sequencing faithfully reflects the advantages of sheep pox virus in binding to PKR compared with eIF2?,and also indicates that GPV may be more infectious than SPV.The key amino acids leading to the interaction differences were identified through the analysis of the complexes.It was found that the 27 and 49 amino acids in the sheep pox virus K3 L gene had an important effect on the function of K3 L.However,the specific mechanism of K3 L in the host range of sheep pox virus and immune escape against host immunity needs further study.This study would be of great significance in elucidating the molecular mechanism of host tropism of the virus,revealing the relationship between antiviral and antiviral inhibition between host and poxvirus,and creating antiviral drugs and vaccines without species differences.