Preliminary Study On The Mechanism Of Porcine Reproductive And Respiratory Syndrome Virus Nsp4 Inhibiting MHC Class Ⅱ Expression

Porcine reproductive and respiratory syndrome（PRRS）is one of the most serious diseases that threatens the health of swine and causes huge economic losses to the swine industry in China and worldwide.The pathogen of this disease is porcine reproductive and respiratory syndrome virus（PRRSV）.Exploring the pathogenic mechanism of PRRSV could provide theoretical basis for the prevention and control of this disease.It was reported that the expression of MHC class Ⅱ protein in cells and on cell surface is interfered by PRRSV infection in vitro.The following studies should be done to explore the key proteins in PRRSV affecting the expression of MHC class Ⅱ,the change of MHC class Ⅱ expression at the m RNA and protein level,and the mechanism of this effect.1.Screening of PRRSV key proteins affecting MHC class Ⅱ expressionFive PRRSV proteins were overexpressed in passageable porcine alveolar macrophages cell line（3D4/21）.Samples were collected at 12 h,18 h,and 24 h after transfection.The changes of SLA-DR expression in total protein and on cell surface of 3D4/21 cells were measured using Western blotting and fluorescence activated cell sorter（FACS）,respectively.Western blotting showed that SLA-DR expression,compared with the control transfected with empty vector,was significantly reduced in total protein after overexpression of PRRSV non-structural protein 4（Nsp4）in 3D4/21 cells for 24 fh.FACS results showed that the expression of SLA-DR on cell surface of 3D4/21 cells decreased from 19.3%to 4.46%after overexpression of PRRSV-Nsp4for 24 h,which was about 4 times lower than control transfected with empty vector which validate the results of Western blotting.These results showed that SLA-DR expression in total proteins and cell surface proteins was significantly reduced under the influence of PRRSV-Nsp4.Therefore,we found that PRRSV-Nsp4 is a key protein of PRRSV that inhibits the expression of MHC class Ⅱ.2.Construction and identification of recombinant lentivirus rLV-Nsp4The PRRSV-Nsp4 gene was amplified and double-digested with EcoR I and Not I to produce cohesive ends.Then it was ligated to the lentivirus expression vector pCD513B.The constructed overexpression plasmid pCD513B-Flag-Nsp4 was identified by double enzyme digestion and DNA sequencing.The expression of Nsp4 protein was detected by Western blotting.HEK-293T cells were co-transfected with pCD513B-Flag-Nsp4 and the third generation lentiviral packaging system helper plasmids（p LP1,p LP2,p LP/VSVG）.The recombinant lentivirus（rLV-Nsp4）was successfully packaged.It was identified by RT-PCR and Western blotting that rLV-Nsp4 stably express PRRSV-Nsp4 in HEK-293T cells.Its TCID₅₀was10^5.66/mL determined by green fluorescent protein（GFP）and Reed-Muench method.This part of the research lays a foundation for further exploring the mechanism of PRRSV-Nsp4 inhibiting MHC class Ⅱ expression in THP-1 cells.3.Preliminary study on the mechanism of PPRRSV-Nsp4 inhibiting MHC class Ⅱ expressionTHP-1 cells were stimulated for 36h by 300 U/mL hIFN-γafter infected with recombinant lentivirus rLV-Nsp4 for 36 h.The infection efficiency of rLV-Nsp4 and its effect on the expression of MHC class Ⅱ and the signaling pathway node molecules of MHC class Ⅱ in THP-1cells was determined by qPCR and Western blotting.The qPCR result showed that in the presence of hIFN-γ,empty lentivirus could upregulate the m RNA levels of MHC class Ⅱ and its signaling pathway node molecules（CⅡTA,STAT1,and IRF1）in THP-1 cells,whereas recombinant lentivirus rLV-Nsp4 was able to inhibit this upregulation.Western blotting analysis showed that recombinant lentivirus rLV-Nsp4 was able to inhibit the expression of phosphorylated STAT1（p-STAT1）and IRF1,which in turn inhibited MHC class Ⅱ expression.In summary,we identified PRRSV-Nsp4 as a key protein in PRRSV that inhibited MHC class Ⅱ expression.A recombinant lentivirus rLV-Nsp4 expressing PRRSV-Nsp4 was successfully constructed.We found that the recombinant lentiviral rLV-Nsp4 inhibited hIFN-γinduced upregulation of MHC class Ⅱ and its signaling pathway node molecules（CⅡTA,STAT1,and IRF1）at m RNA levels.Moreover,the recombinant lentivirus rLV-Nsp4 reduced the intracellular MHC class Ⅱ expression at both m RNA and protein levels by inhibiting the protein expression of p-STAT1 and IRF1.Our findings initially reveales the mechanism that PRRSV reduced antigen presentation ability of antigen-presenting cells to evade cellular immune responses after infecting,which deepen the understanding of PRRSV infection and provide an important theoretical basis for PRRS prevention and control.