Screening And Functional Identification Of PRV Proteins Affecting TAP Peptide Transport

Pseudorabies virus(PRV)belongs to Alphaherpesvirinae subfamily of herpesviridae.Swine is its natural host.PRV can cause abortion of pregnant sows,infertility of breeding swines,diarrhea of newborn piglets and nervous system symptoms,which brings huge economic losses to the swine industry in the world.The virus can proliferate in swines and establish long-term latent infection in the peripheral nervous system.When the external environment changes or the immunity of swines is low,the state of latent infection can be reactivated to the state of acute infection,resulting in re-infection,which brings difficulties to the prevention and treatment of PR.The endogenous antigens absorbed by antigen presenting cells are degraded into polypeptides under the action of proteasome,which are bound to the cytoplasmic side antigen binding of antigen processing related transporters on the endoplasmic reticulum to activate ATP enzyme on TAP.ATP hydrolysis releases energy to change the structure of TAP dimer,and the transmembrane channel is opened,and the antigenic peptide is then transported to the endoplasmic reticulum cavity.Antigen peptides bind to MHCI molecules to form a peptide MHC-complex,which is then displayed on the cell surface.Cytotoxic T lymphocytes play a cytotoxic role by recognizing the complexes on the cell surface and clearing the infected cells.Peptide loading complex(PLC)is composed of TAP,Tapasin,calcitonin,calreticulin and ERp.TAP heterodimer composed of TAP1 and TAP2 is located in the center of PLC.It is the transferring channel of polypeptides from cytoplasm to endoplasmic reticulum and plays a decisive role in the whole process of antigen presentation.Tapasin is an important bridge between TAP and the rest of PLC.Its transmembrane domain binds to TAP1 and stabilizes the structure of TAP.Therefore,the absence of TAP and Tapasin may cause the peptides that bind to TAP can not bind MHC I molecules effectively and present to the cell surface.It has been confirmed that PRV can induce immune escape by down-regulating the expression of swine leukocyte antigen class I molecules(SLA I,porcine MHC class I molecules)on the host cell surface,but the molecular mechanism of down-regulation of SLA I expression has not been fully elucidated.Inhibition of the transport of TAP antigenic peptides is one of the important factors leading to the down-regulation of MHC I expression.Therefore,in this study,bioinformatics methods were used to screen PRV proteins that affect the transport of TAP peptides,and their functions were verified.1.Preliminary screening of pseudorabies virus proteins affecting TAP peptide transport by bioinformaticsThe five known TAP inhibitors such as HSV-1 ICP47,BHV-1 UL49.5,HCMV US6,EBV BNLF2a and CPXV CPXV012 were analyzed by on-line analysis software.PRV proteins similar to several TAP inhibitors were screened,and the homology and tertiary structure of TAP inhibitors and selected PRV proteins were predicted and compared.The hydrophobic protein PRV pUL49.5 with one signal peptide,two transmembrane domains and mainly located in endoplasmic reticulum,which is the same as TAP inhibitor BHV-1 UL49.5,as well as the hydrophilic proteins PRV pus6 and PRV pul44 with one signal peptide,one transmembrane domain and are mainly located in plasma membrane and endoplasmic reticulum,which have the same structure as HCMV US6,were screened.2.Construction and expression of eukaryotic expression recombinant plasmid of pseudorabies virus US6 and UL44 genesEukaryotic expressing plasmids pUS6-HA and pUL44-HA,were constructed and expressed in PK-15 cells by conventional liposome transfection.The effects of pUS6 and pUL44 on total SLA I molecule and cell surface SLA I molecule were analyzed by qRT-PCR and Western blot.The results showed that US6 and UL44 could significantly down-regulate the total SLA I molecule at the mRNA and protein level and downregulate the surface SLA I molecule at the protein level,suggesting that the candidate protein may affect the host immune response by regulating the expression of SLA I itself,inhibiting TAP presenting antigenic peptide,making SLA I detained in endoplasmic reticulum,transferring newly assembled SLA I into lysosome or promoting cell surface SLA I endocytosis into lysosome.3.Functional Identification of pseudorabies virus pUS6 and pUL44 affecting TAP Peptide TransportTo investigate whether the observed down-regulation of SLA I on the cell surface is dependent on the inhibition of PLC,qRT-PCR and Western blot were used to detect the effects of TAP and Tapasin,CNX,CRT,ERp,CRT and other molecules after expressing candidate proteins.The results showed that pUS6 inhibited the transcription of TAP1,TAP2 and Tapasin molecules in a dose-independent manner.In order to further verify the effect of pUS6 on TAP,the binding activity of TAP to ATP was detected by ATP-agarose gel binding assay.The results verified by Western blot showed that PRV pUS6 could inhibit the binding of TAP and ATP.In summary,PRV pUS6 may inhibit the binding activity of ATP and TAP to make the transfer of antigen peptides lack of energy,causing the peptides bound to TAP to be unable to bind to SLA I molecules to form a complex,and ultimately cause the synthesis of TAP and Tapasin to be blocked and affect SLA I on the cell surface.PRV pUL44 down-regulates SLA I molecules on cell surface,and the mechanism involved in virus immune escape is not clear,which needs to be further studied.These findings do not only highlight the functions of PRV-encoded protein in viral immune escape,but provide new insights into the interactions between viral infections and their hosts...