The Interaction Of Duck Plague Virus Glycoproteins I And E And Their Effects On Viral Replication And Virulence

To explore the functions of duck plague virus(DPV)glycoproteins I and E on viral basic biological functions,single-gene deletion strains and a double-genes deletion strain as well as their revertant viruses,named BAC-CHv-?gI,BAC-CHv-?gE,BAC-CHv-?gI+?gE,BAC-CHv-?gI Rev,BAC-CHv-?gE Rev and BAC-CHv-?gI+?gE Rev,were constructed by using a markerless two-step Red recombination system implemented on DPV CHv genome cloned into a bacterial artificial chromosome(BAC-CHv).The interaction of gI and gE,as well as their effects on DPV replication,including viral assembly,release and cell-cell spread,as well as viral virulence,were carried out on this study and the main results are listed as follows.1.The interaction of DPV gI and gE and their effects on viral assembly,release and cell-cell spreadResults in this paper confirmed that DPV gI and gE formed a complex.It was also found that the molecular size of gE was affected by gI.Viral growth curve determined that DPV gI/gE complex not only participated in the formation of virions in cells but also promoted the release of virions into cell supernatant.However,when expressed gI or gE proteins alone,the formation of virions in cells,as well as the release of virions into cell supernatant,were inhibited.Transmission electron microscopy assay confirmed that the gI/gE complex played an important role in viral final envelopment but the single expression of gI or gE could not promote viral final envelopment.Viral plaque assay confirmed that DPV gI/gE complex promoted viral cell-cell spread,however,when expressed gI or gE protein alone,only a few numbers of viruses could spread between cells.2.The effect of gI protein on viral cell-cell spread and viral secondary envelopment of DPV2.1 The change of the localization and the expression of gE protein after the infection of CHv-?gIThis paper constructed a recombinant virus without the expression of EGFP green fluorescent and gI,named CHv-?gI.By using indirect immunofluorescence assay,it could be confirmed that DPV gI promoted gE located from the endoplasmic reticulum(ER)to the Golgi.By using Western blot,indirect immunofluorescence assay,as well as the sugar chain digestion experiment,it could be concluded that the change of the molecular size of gE protein was caused by the assistance of gI protein to promote gE protein locate at Golgi and then occurred O-glycosylation at Golgi.2.2 Selection of the key amino acid domains of gI protein that affected the localization of gE protein at GolgiGolgi was an important site for viral cell-cell spread and viral final envelopment processes.The gE failed to locate at the Golgi after deleting gI could be the main reason for the inability of the single gE to participate in viral cell-cell spread and viral final envelopment processes.Eukaryotic expression plasmids containing N-terminal of extracellular domain,C-terminal of extracellular domain,transmembrane domain as well as intracellular domain were constructed at amino acids 1-82,1-106,1-158,1-279,1-302 and1-317 of gI protein.By using indirect immunofluorescence assay,it could be confirmed that1-158aa of the N-terminal of extracellular domain of gI protein could retain gE protein located at the ER and 1-279aa of the C-terminal of extracellular domain of gI protein could assist gE located at the Golgi.BAC-CHv-?gI(159-371)and BAC-CHv-?gI(280-371)were successfully constructed.By using indirect immunofluorescence assay,the result further confirmed that the N-terminal 1-158 aa of extracellular domain of gI protein retained gE protein at the ER,while the 1-279 aa of the gI protein promoted gE locate to the Golgi.2.3 Determine the reason why gI protein promoted gE protein locate from the ER to the GolgiIP test confirmed that 1-158aa and 1-279aa of the extracellular domain of gI protein could interact with gE protein.According to the phenomenon of misfolded protein retained in the ER by proofreading system,it could be seen that the N-terminal 1-158aa of extracellular domain of gI protein formed a misfolded complex with gE protein and then retained in the ER,however,the C-terminal 1-279aa of extracellular domain of gI protein formed a correctly folded complex with gE protein and then released from the ER.That is,only after gI protein and gE protein form a correctly folded complex that could be recognized by the ER protein proofreading system,can the proteins be released from the ER and localized to the Golgi.3.The effect of gI protein on the virulence of DPVTen 28-day-old ducks were infected with CHv-?gI and CHv-50 by 10³,10⁴,10⁵ and10⁶ TCID₅₀,respectively.The clinical symptoms,pathological changes and viral load of each organ were compared.The results showed that:(1)compared with the CHv-50 parent strain,CHv-?gI of each infection dose did not cause the increase of body temperature of ducks,death of ducks,and clinical pathological changes of tissues and organs of ducks.However,the body temperature of ducks increased to 43?after CHv-50 parent strain infection.It indicated that the virulence of DPV was decreased after the deletion of gI gene.(2)QPCR was used to detect the genome copies of different tissues and organs in ducks infected with CHv-50 and CHv-?gI.The results showed that when compared with CHv-50,the average genome copies of immune organs and parenchymal organs in CHv-?gI-infected ducks were maintained about 10⁴copies/g on the 1st-9th day.However,after CHv-50infection,the average genome copy number of immune organs and parenchymal organs increased from 10⁴ copies/g to 10⁵-10⁹ copies/g.It indicated that the inhibition of replication ability of CHv-?gI in immune organs and parenchymal organs of ducks was one of the main reasons why the virulence of DPV attenuated after deletion of gI.Above all,DPV gI and gE proteins interact with each other in the process of viral formation,and the formed gI/gE complex could promote viral cell-cell spread,as well as promote the formation of complete virus particles through promoting viral assembly by participating in the process of viral secondary envelopment and promote the release of complete virus particles into the cell supernatant.gI and gE proteins could not independently complete the above processes.It was also found that only after gI protein and gE protein form a correctly folded complex that could be recognized by the ER protein proofreading system,can the proteins be released from the ER and localized to the Golgi where the O-glycosylation of gE as well as viral cell-cell spread and viral final envelopment could occur.