Molecular Mechanism Of Host SLA-? Downregolation By Pseudorabies Virus In Vitro

Pseudorabies virus(PRV)is the causative agent of pseudorabies(PR)which causes large economic losses for swine industry of the world.Immune escape is an important characteristic of PRV infection which results in persistent infection and difficulty for PRV eradication.Previous studies confirmed that PRV could escape host immune response through downregulation of porcine MHC-?(Swine leukocyte antigen class ?,SLA-?)molecules on infected cells.But the molecular mechanism of SLA-? downregulation is not clear.The aim of this study is to figure out the mechanism of how PRV down-regulates SLA-? molecules using the PRV variant HB1201 and identify the viral protein(s)involved in this downregulation to provide the scientific evidence for elucidating pathogenic mechanism of PRV.To examine the effect of PRV on cell surface SLA-?,the level of surface SLA-? on PRV variant HB1201 infected porcine kidney(PK-15)cells was analyzed by the flow cytometry.The results showed that PRV could down-regulate surface SLA-? levels of PK-15 cells.Moreover,the effects of PRV on cellular SLA-? heavy chain(HC)and ?2 microglobulin(?2m)of PK-15,porcine alveolar macrophages(PAMs)and swine testis(ST)cells were analyzed by western blot.The results showed that PRV significantly down-regulated SLA-? HC,but not ?2m,in these three infected cells.Meanwhile,this study found that SLA-? HC downregulation by PRV was dependent on viral replication and that the ability of SLA-? HC downregulation between PRV variant HB1201 and classical strain Fa is no obvious difference.The downregulation of SLA-? HC was markedly inhibited by the treatment of lysosome inhibitor NH4Cl and CQ,suggesting that PRV mainly down-regulates SLA-? HC through lysosome pathway.To identify the viral protein(s)that was(were)essential for SLA-? HC downregulation,six candidate proteins(pUL56,EP0,pUL41,gE,pUL49.5 and pUS3)were selected according to analysis and prediction of their functions by bioinformatics.The effects of these proteins on SLA-? HC in cotransfected cells were analyzed by western blot.The results showed that PRV pUL56,except for pUS3 whose downregulation for SLA-? has been confirmed,was able to down-regulate SLA-? HC.Moreover,the deletion of UL56 gene led to the decreased ability to down-regulate SLA-? HC,suggesting that pUL56 is essential for SLA-? HC downregulation by PRV.To analyze the pathway of SLA-? HC downregulation by pUL56,the proteasome inhibitor MG132,lysosome inhibitor NH4Cl and CQ,ubiquitin activating enzyme E1 inhibitor PYR41 were used to treat transfected cells and then SLA-? HC level was examined by western blot.The results showed that the degradation of SLA-? HC was inhibited by the treatment of NH4Cl,CQ and PYR41,suggesting that pUL56 mainly down-regulates SLA-? HC through lysosome pathway which is consistent with SLA-? HC downregulation pathway by PRV.Mutate the four PPXY motifs to AAXY elements to analyze their functions in SLA-? HC degradation.Western blot results showed that the ability of pUL56 AY to degrade SLA-? HC became weaker compared with that of pUL56 WT,demonstrating that PPXY motifs are essential for the degradation of SLA-? HC by pUL56.Meanwhile,Co-IP and confocal microscope results confirmed that pUL56 and SLA-? HC interacted with each other not only in transfected cells but in infected cells.Moreover,ubiquitination assay results showed that pUL56 increased ubiquitination levels of SLA-? HC,in which PPXY motifs played important roles.In conclusion,our studies demonstrated that PRV down-regulated SLA-? HC through lysosome pathway,resulting in the decrease of surface SLA-? molecules.PRV pUL56 increased the ubiquitination levels of SLA-? HC to degrade it through lysosome pathway,in which PPXY motifs played important roles.This research reveals a molecular mechanism of SLA-? downregulation by PRV,which provides the scientific evidence for elucidating pathogenic mechanism of PRV.