TRIM37对猪繁殖与呼吸综合征病毒复制及IFN-β产生影响的研究

Study on the effect of TRIM37 on PRRSV replication and IFN-βproduction

本实验室前期通过质谱分析筛选到宿主因子三方基序蛋白37(TRIM37),推测其可能与猪繁殖与呼吸综合征病毒(PRRSV)的复制相关。为探究TRIM37对PRRSV复制的影响,本研究将不同剂量的PRRSV HN07-1株(MOI分别为0.01、0.1、1)分别感染猪肺泡巨噬细胞(PAM)和MARC-145细胞后不同时间(6 h、12 h、24 h、36 h和48 h),采用荧光定量PCR(qPCR)检测细胞中内源TRIM37的转录水平,结果显示PRRSV能够刺激这两种细胞内源TRIM37的转录,并且PRRSV以MOI 1及感染后24 h细胞中TRIM37的转录水平最高,感染后36 h和48 h细胞中TRIM37的转录水平较感染后24 h有所下降但仍显著高于对照组(P<0.05)。从MARC-145细胞中经PCR扩增TRIM37基因并克隆至pCAGGS-HA中构建真核表达质粒pCAGGS-HA-TRIM37,经双酶切及测序鉴定正确后转染MARC-145细胞,24 h后以MOI 1将HN07-1株感染细胞,感染后不同时间分别采用qPCR、western blot、病毒滴度(TCID50)测定和间接免疫荧光试验(IFA)检测PRRSV的复制情况。结果显示,与转染空载体pCAGGS-HA的对照细胞相比,过表达TRIM37后细胞中PRRSV ORF7的转录水平、N蛋白的表达水平(感染后48 h和72 h)及病毒滴度(感染后36 h与48 h)均显著(P<0.05)或者极显著(P<0.01)下降。针对TRIM37基因设计3对特异性的TRIM37 siRNA-1/2/3,转染MARC-145细胞后,利用qPCR检测TRIM37 siRNA的干扰效率,结果显示,TRIM37 siRNA-2能够有效降低TRIM37的转录水平。将TRIM37 siRNA-2转染MARC-145细胞后再以MOI 1 HN07-1株感染,感染后不同时间分别采用qPCR、western blot和病毒滴度(TCID50)测定检测PRRSV的复制情况。结果显示,与转染NC siRNA的对照细胞相比,干扰TRIM37表达的细胞中PRRSV ORF7的转录水平、N蛋白的表达水平及病毒滴度均显著(P<0.05)或极显著(P<0.01)升高。进一步将不同剂量的pCAGGS-HA-TRIM37与报告质粒pRL-TK-Luc和pIFN-β-Luc共转染HEK293T细胞;同时将不同剂量的pCAGGS-HA-TRIM37与报告质粒pRL-TK-Luc和pISRE-Luc共转染HEK293T细胞,18 h后加入poly(I:C)处理上述各组细胞,6 h后利用双荧光素酶试剂盒检测各组细胞中IFN-β和ISRE启动子的活性。将pCAGGS-HA-TRIM37转染HEK293T细胞,18 h后加入poly(I:C)处理细胞,6 h后采用qPCR检测细胞中相关干扰素刺激基因(ISG)(ISG15、CXCL10、MX1和OAS1)相对转录水平。双荧光素酶试验结果显示,与转染空载体的对照细胞相比,过表达TRIM37显著上调细胞中poly(I:C)激活的IFN-β和ISRE启动子的活性(P<0.05),且这种上调作用具有剂量依赖性。qPCR结果显示,与转染空载体的对照细胞相比,过表达TRIM37显著上调细胞中poly(I:C)激活的ISG15、CXCL10、MX1和OAS1 mRNA的转录水平(P<0.05)。本研究首次表明TRIM37能够通过促进细胞中相应干扰素的表达抑制PRRSV复制,该结果丰富了病毒感染过程中PRRSV-宿主相互作用的网络和机制,深化了对PRRSV致病机制的认知。

The host factor TRIM37 was screened through mass spectrometry analysis in our laboratory and it was speculated to be related to the replication of porcine reproductive respiratory syndrome virus(PRRSV)infection.To explore the effect of TRIM37 on PRRSV replication,porcine alveolar macrophages(PAM)and MARC-145 cells were infected with PRRSV HN07-1 strain at a MOI of 0.01,0.1 and 1 for 6,12,24,36,or 48 hours,respectively,and the endogenous mRNA transcription level of TRIM37 was detected by qPCR.The results showed that the transcription of endogenous TRIM37 could be stimulated by PRRSV on these two kinds of cells.The transcription levels of TRIM37 were the highest at MOI 1 for 24 hours post-infection(hpi)while they were decreased at 36 hpi and 48 hpi compared with those at 24 hpi,but still obviously higher than that in the control group(P<0.05).TRIM37 gene was amplified by PCR from MARC-145 cells and cloned into pCAGGS-HA to construct the eukaryotic expression plasmid pcAGGS-HA-TRIM37.After identification by enzyme digestion analysis and sequencing,the resulting plasmid was transfected into MARC-145 cells for 24 hours,and PRRSV strain HN07-1 was infected at MOI 1.The replication level of PRRSV was detected by qPCR,western blot,virus titer(TCID50)and indirect immunofluorescence assay(IFA)at different time points after infection.The results showed that the transcription level of PRRSV ORF7,the expression level of N protein(48 and 72 hpi)and the viral titers(36 and 48 hpi)in the cells with the overexpression of TRIM37 were significantly(P<0.05)or extremely significantly(P<0.01)decreased compared with those in the control cells transfected with empty vector pCAGGS-HA.Three TRIM 37 siRNAs(siTRIM37-1/2/3)were designed for TRIM37 gene,and the knockdown efficiencies of siTRIM37s were detected by qPCR.The results showed that the transcription level of TRIM37 was reduced effectively by siTRIM37-2.MARC-145 cells were transfected with siTRIM37-2 and then infected with HN07-1 strain at MOI 1.The replication level of PRRSV was detected by qPCR and western blot and virus titer(TCID50)at different time points after infection.The results showed that PRRSV ORF7 transcription level,N protein expression level and viral titers in TRIM37 knockdown cells were significantly(P<0.05)or extremely significantly(P<0.01)higher than those in the control cells transfected with NC siRNA.To explore the role of TRIM37 in innate immune response,different doses of pCAGGS-HA-TRIM37 and reporter plasmids of pRL-TK-Luc and pIFN-β-Luc were co-transfected into HEK293T cells.At the same time,different doses of pCAGGS-HA-TRIM37 and reporter plasmids of pRL-TK-Luc and pISRE-Luc were co-transfected into HEK293T cells.After 18 hours,poly(I:C)was added to stimulate the cells in the above groups.After 6 hours,the activities of IFN-βand ISRE promoter in each group were detected by dual luciferase assay.The plasmid of pCAGGS-HA-TRIM37 was also transfected into HEK293T cells,and,the cells were stimulated with poly(I:C)post transfection of 18 hours.After 6 hours,qPCR was used to detect the relative transcription levels of interferon stimulated genes(ISG15,CXCL10,MX1 and OAS1)in the cells.The results of dual luciferase assay showed that TRIM37 overexpression significantly up-regulated the activities of IFN-βand ISRE promoter activated poly(I:C)in the cells compared with the control cells transfected with the empty vector(P<0.05),and this up-regulation effect was in dose dependent.The results of qPCR showed that TRIM37 overexpression significantly up-regulated the transcription levels of ISG15,CXCL10,MX1 and OAS1 mRNA activated by poly(I:C)in the cells compared with the control cells transfected with the empty vector(P<0.05).These results indicated that TRIM37 overexpression could promote the expression of related interferons in host cells.This study demonstrated for the first time that TRIM37 could inhibit PRRSV replication by promoting the expression of interferons and corresponding ISG in cells,which enriched the network and mechanism of PRRSV-host interaction during viral infection and deepened the understanding of the pathogenic mechanism for PRRSV.