抗伪狂犬病病毒干扰素刺激基因的筛选及IFIT3抗病毒活性的鉴定
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摘要
为研究抗伪狂犬病病毒(PRV)特异性的干扰素刺激基因(ISGs),本研究采用与表达增强型绿色荧光蛋白报告病毒相结合的高内涵筛选系统对具有抗病毒功能的10种ISGs进行筛选,之后利用过表达细胞系、间接免疫荧光(IFA)和q RT-PCR试验对显著抑制PRV复制的IFIT3 (p<0.01)进行抗病毒活性验证,通过q RT-PCR检测了PRV感染的猪肺泡巨噬细胞(PAM)和PRV感染猪的外周血单核细胞(PBMC)中IFIT3 m RNA的转录水平。高内涵筛选结果显示,5种潜在的抗PRV ISGs分子(IFIT3、ISG15、GBP2、Mx1和ISG20)对PRV的复制具有显著的抑制作用,其中IFIT3效果最显著;IFA和q RT-PCR试验结果显示,在PK-15细胞和PAM中,过表达IFIT3均可以显著降低PRV-TJ株的病毒滴度和基因组拷贝数;q RT-PCR试验结果显示,在PRV感染的PAM和PBMC中,IFIT3 m RNA的转录水平均显著上调。流式细胞术试验表明过表达IFIT3不影响PK-15细胞凋亡,推测IFIT3可能存在其它的抗病毒机制。本研究筛选到5种潜在的抗PRV ISGs并证实IFIT3是一种抗PRV的ISG,为抗PRV ISGs的相关研究,特别是猪源IFIT3抗病毒机制的研究奠定了基础。
In this study, a high-content screening combined with a reporter pseudorabies virus expressing enhanced green fluorescent protein(rPRV-EGFP) was used to screen 10 kinds of interferon-stimulated genes(ISGs) and explore whether they have anti-PRV function. ISGs were screened, and then the overexpressing cell line, IFA and qRT-PCR were used to verify the antiviral activity of the IFN-induced protein with tetratricopeptide repeats 3(IFIT3), which had the most significant inhibitory effect on PRV replication. In addition, the transcription levels of IFIT3 m RNA in PRV-infected porcine alveolar macrophage cells(PAM) and in peripheral blood mononuclear cells(PBMC) of PRV-infected pigs were detected by qRT-PCR. High-content screening results showed that five potential anti-PRV ISGs(IFIT3, ISG15, GBP2, Mx1 and ISG20) had significant inhibitory effects on PRV replication, and IFIT3 had the most significant inhibitory effect on PRV replication. The results of IFA and qRT-PCR showed that overexpression of IFIT3 in PK-15 and PAM significantly reduced the viral titer and genome copy number of PRV-TJ strain.qRT-PCR assay showed that the transcription level of IFIT3 mR NA was significantly up-regulated in PRV-infected PAM and PBMC. In terms of the molecular mechanism of IFIT3 against PRV, flow cytometry test showed that overexpression of IFIT3 did not affect apoptosis, suggesting that there may be other antiviral mechanisms of IFIT3. This study has successfully screened 5 kinds of potential anti-PRV ISGs and confirmed the IFIT3 has inhibitory effects on PRV replication, laid a foundation for the further study of anti-PRV ISGs, especially on the antiviral mechanism of porcine IFIT3.
In this study, a high-content screening combined with a reporter pseudorabies virus expressing enhanced green fluorescent protein(rPRV-EGFP) was used to screen 10 kinds of interferon-stimulated genes(ISGs) and explore whether they have anti-PRV function. ISGs were screened, and then the overexpressing cell line, IFA and qRT-PCR were used to verify the antiviral activity of the IFN-induced protein with tetratricopeptide repeats 3(IFIT3), which had the most significant inhibitory effect on PRV replication. In addition, the transcription levels of IFIT3 m RNA in PRV-infected porcine alveolar macrophage cells(PAM) and in peripheral blood mononuclear cells(PBMC) of PRV-infected pigs were detected by qRT-PCR. High-content screening results showed that five potential anti-PRV ISGs(IFIT3, ISG15, GBP2, Mx1 and ISG20) had significant inhibitory effects on PRV replication, and IFIT3 had the most significant inhibitory effect on PRV replication. The results of IFA and qRT-PCR showed that overexpression of IFIT3 in PK-15 and PAM significantly reduced the viral titer and genome copy number of PRV-TJ strain.qRT-PCR assay showed that the transcription level of IFIT3 mR NA was significantly up-regulated in PRV-infected PAM and PBMC. In terms of the molecular mechanism of IFIT3 against PRV, flow cytometry test showed that overexpression of IFIT3 did not affect apoptosis, suggesting that there may be other antiviral mechanisms of IFIT3. This study has successfully screened 5 kinds of potential anti-PRV ISGs and confirmed the IFIT3 has inhibitory effects on PRV replication, laid a foundation for the further study of anti-PRV ISGs, especially on the antiviral mechanism of porcine IFIT3.
引文
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[15]Abbas Y M,Pichlmair A,Gorna M W,et al.Structural basis for viral 5'-PPP-RNA recognition by human IFIT proteins[J].Nature,2013,494(7435):60-64.
[16]Yang Ying-yun,Zhou Ye,Hou Jin,et al.Hepatic IFIT3 predicts interferon-αtherapeutic response in patients of hepatocellular carcinoma[J].Hepatology,2017,66(1):152-166.
[17]Liu Xin-yi,Chen Wei,Wei Bo,et al.IFN-induced TPR protein IFIT3 potentiates antiviral signaling by bridging MAVS and TBK1[J].J Immunol,2011,187(5):2559-2268.
[2]Hagen C,Dent K C,Zeev-Ben-Mordehai T,et al.Structural basis of vesicle formation at the inner nuclear membrane[J].Cell,2015,163(7):1692-1701.
[3]Ivashkiv L B,Donlin L T.Regulation of type I interferon responses[J].Nat Rev Immunol,2014,14(1):36-49.
[4]Su Chen-he.Herpes simplex virus 1 abrogates the cG AS/STING-mediated cytosolic DNA-Sensing pathway via its virion host shutoff protein,UL41[J].J Virol,2017,91(6):e02414-16.
[5]Jiang Zhang-tao,Su Chen-he,Zheng Chun-fu.Herpes simplex virus 1 tegument protein UL41 counteracts IFIT3 antiviral innate immunity[J].J Virol,2016,90(24):11056-11061.
[6]Wang Ji-chun,Guo Rong-li.An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant[J].BMC Vet Res,2016,12(1):277.
[7]Borden E C,Williams B R.Interferon-stimulated genes and their protein products:what and how?[J].J Interferon Cytokine Res,2011,31(1):1-4.
[8]Sadler A J,Williams B R.Interferon-inducible antiviral effectors [J].Nat Rev Immunol,2008,8(7):559-568.
[9]Reed L M H.A simple method of estimating 50 percent endpoints[J].Am J Hyg,1938,27:493-497.
[10]Meng Xing-yu,Luo Yu-zi,Liu Yan,et al.A triplex real-time PCR for differential detection of classical,variant and BarthaK61 vaccine strains of pseudorabies virus[J].Arch Virol,2016,161(9):2425-2430.
[11]Zhou Xiang,Michal J J,Zhang Li-fan,et al.Interferon induced IFIT family genes in host antiviral defense[J].Int J Biol Sci,2013,9(2):200-208.
[12]Hsu Y L,Shi Shao-fu,Wu Wan-li,et al.Protective roles of interferon-induced protein with tetratricopeptide repeats 3(IFIT3)in dengue virus infection of human lung epithelial cells[J].PLoS One,2013,8(11):e79518.
[13]Zhang Li-li,Liu Jie,Bai Juan,et al.Poly(I:C)inhibits porcine reproductive and respiratory syndrome virus replication in MARC-145 cells via activation of IFIT3[J].Antiviral Res,2013,99(3):197-206.
[14]Niess H,Camaj P,Mair R,et al.Overexpression of IFN-induced protein with tetratricopeptide repeats 3(IFIT3)in pancreatic cancer:cellular"pseudoinflammation"contributing to an aggressive phenotype[J].Oncotarget,2015,6(5):3306-3318.
[15]Abbas Y M,Pichlmair A,Gorna M W,et al.Structural basis for viral 5'-PPP-RNA recognition by human IFIT proteins[J].Nature,2013,494(7435):60-64.
[16]Yang Ying-yun,Zhou Ye,Hou Jin,et al.Hepatic IFIT3 predicts interferon-αtherapeutic response in patients of hepatocellular carcinoma[J].Hepatology,2017,66(1):152-166.
[17]Liu Xin-yi,Chen Wei,Wei Bo,et al.IFN-induced TPR protein IFIT3 potentiates antiviral signaling by bridging MAVS and TBK1[J].J Immunol,2011,187(5):2559-2268.