Mechanism And Significance Of Inhibition Of B2M Gene Expression By Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome is caused by porcine reproductive and respiratory syndrome virus infection,mainly represents reproductive failure of sows and respiratory disease of piglets,the disease has immunosuppression and immune escape features,it also has a variety of strategies to evade and inhibit the immune clearance of the host,resulting in persistent infection.Major histocompatibility complex Ⅰ(MHC-Ⅰ)is present in the surface of all nucleated cells,mainly involved in the presentation of endogenous antigens,especially viral antigens.The MHC-Ⅰ antigen presentation process present the viral antigens to the surface of infected cells,so as to be recognized by CD8 + T lymphocytes,activation of CD8 + cytotoxic T lymphocytes can effectively remove infected cells,to complete the clearance of the virus.Swine leukocyte antigen Ⅰ(SLA-Ⅰ)is the Major histocompatibility complex Ⅰin pigs,is constituted by heavy chain and β2m,β2m which maintain MHC-Ⅰstructural stability,and play an important role in peptides binding in antigen presentation process.In nature,many viruses have different mechanisms to interfere the presenting of viral antigen to evade the immune clearance fo host.Studies have reported that PRRSV can down-regulate the MHC-Ⅰmolecule expression on the surface of porcine pulmonary alveolar macrophages(PAMs)by increasing the MHC-Ⅰ molecule ubiquitin-proteasome degradation.To further resolve the mechanism how PRRSV down-regulate the surfaces MHC-Ⅰ molecule expression,we carried out this study hope that through more research to provide more scientific explanations about immunosuppression and immune escape mechanisms of PRRSV.First,indirect immunofluorescence assay and flow cytometry analysis showed that PRRSV can lead to the MHC-Ⅰ molecules expression reduction on the surface of antigen presenting cells such as PAMs,bone marrow-derived dendritic cells(BMDCs)and also can decrease the MHCⅠ molecules expression on the surface of Marc145 cells infected by PRRSV.Further,we mainly used highly pathogenic PRRSV strains and Marc145 cells as our reserch materials,and we found that the protein levels of both MHC-Ⅰ and β2m were reduced by western blot with the monoclonal antibody against MHC-Ⅰ-HC and β2m,respectively.The results showed that PRRSV infection could significantly down-regulate the expression of MHC-Ⅰ and β2m in Marc145 cells.After using ubiquitin E1 enzyme inhibitor PYR41 to inhibit protein ubiquitin degradation,the protein levels of β2m still could not recover,and realtime qPCR results also showed that PRRSV can reduce the mRNA levels ofβ2m in Marc145 and PAMs,it meaned that PRRSV can inhibit the transcription level of β2m.Then,constructed various eukaryotic expression vectors which could express the structural proteins and non-structural proteins of PRRSV.Then transfected Marc145 and PK-15 cells and anylyzed by flow cytometry.The results showed that the tranfection of viral non-structural protein 4(Nsp4)reduced the MHC-Ⅰ molecules exprssion on Marc145 cell surface and also on PK-15 cell surface,while both the transcription level of β2m mRNA and the protein level of β2m is declined.The results of Dual luciferase reporter gene system test indicated that Nsp4 can inhibit the transcription of β2m.ChIP-PCR results also proved that,Nsp4 were combined with β2m promoterregulatory region sequence β2m to regulate the transcription of β2m.In summary,our study showed that PRRSV could decrease the β2m transcriptional level to inhibit the expression of β2m protein,which reduced the expression MHC-Ⅰ molecules on cell surface,further affectted the presentation of viral antigens,thereby evaded the recognition of CD8 + cytotoxic T lymphocytes to avoid the elimination by host.Our results indicate a new mechanism impairing the MHC-Ⅰmediated antigen presentation and provide valuable evidence for understanding of immune suppression and immune evasion of PRRSV.