PRRSV Nsp4基因突变株对β2M表达的影响

Effect of PRRSV Nsp4 gene mutant strains on the expression ofβ2M

[目的]本试验旨在探究猪繁殖与呼吸综合征病毒(PRRSV)Nsp4基因是否在抑制细胞β2M表达方面发挥关键性作用,以进一步研究PRRSV对抗原递呈的免疫抑制机制。[方法]建立PRRSV的反向遗传平台并成功拯救出PRRSV野生毒株SH2020。根据之前对Nsp4基因不同结构域的研究以及氨基酸位点功能预测,在其DomainⅡ和DomainⅢ分别筛选出3个和5个不同的氨基酸位点,并在病毒感染性克隆质粒上对这些位点进行不同的氨基酸替换突变,再将构建好的感染性克隆质粒转染Marc-145细胞以拯救各个Nsp4突变PRRSV病毒,将拯救的PRRSV病毒以MOI=0.02感染PAM细胞,并通过RT-qPCR和Western blot验证PRRSV Nsp4基因是否能够在PAM细胞β2M表达方面发挥作用。[结果]成功拯救出PRRSV D2-5(Nsp4-A80L)突变毒株,并且在Marc-145细胞上的生长动力学特性与野生毒株SH2020相似;而Nsp4基因其余位点突变则会导致PRRSV生长受到显著抑制从而无法进行试验。Western blot和RT-qPCR试验结果证实PRRSV D2-5毒株能够显著促进β2M在Marc-145和PAM细胞的表达,而野生毒株SH2020则表现出了对β2M表达的抑制作用。[结论]Nsp4基因在PRRSV抑制细胞β2M表达方面发挥重要作用,并且Nsp4-A80L突变能够显著促进β2M的表达。

[Objectives]This study was conducted to investigate whether porcine reproductive and respiratory syndrome virus(PRRSV)Nsp4 gene played a key role in the inhibition ofβ2M expression,which laid a foundation for further study of the immunosuppressive mechanism of PRRSV on antigen presentation.[Methods]In this experiment,the reverse genetic platform of PRRSV was established,and the wild PRRSV strain SH2020 was successfully saved.Based on the previous research on different domains of Nsp4 gene and the prediction of amino acid site function,three and five different amino acid sites in the DomainⅡand DomainⅢof NSP4 gene were screened,and different amino acid substitution mutations were performed on viral infectious cloning plasmids.The constructed infectious clone plasmids were then transfected into Marc-145 cells to rescue Nsp4 mutant PRRSV viruses.The rescued PRRSV virus was used to infect PAM cells at a MOI of 0.02,and RT-qPCR and Western blot were used to verify whether PRRSV Nsp4 gene could play a role in the expression ofβ2M in PAM cells.[Results]The mutant strain D2-5(Nsp4-A80L)was rescued successfully,the character of growth kinetics of which was similar to that of wild strain SH2020;but other mutant sites of Nsp4 gene led the restriction to the replication of the PRRSV which was not accessible to the experiment assay.It was verified that D2-5 strain could enhance the expression of theβ2M on the Marc-145 and PAM through the Western blot and RT-qPCR while the wild strain SH2020 presented the inhibition to theβ2M.[Conclusions]Nsp4 gene plays an important role in inhibiting the expression ofβ2M molecule in PRRSV cells,and Nsp4-A80L mutation can significantly promote the expression ofβ2M.