泛素特异性蛋白酶18抑制IFNα抗HBV活性但并不抑制IFNλ抗HBV的活性（英文）
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摘要
干扰素α(IFN-α)是临床最常用的抗乙型肝炎病毒(HBV)药物之一。泛素特异性蛋白酶18(USP18)被证实是抑制IFN-α抗HBV活性的因子,但USP18是否对干扰素λ(IFN-λ)抗HBV有影响还尚未可知。为了明确USP18对IFNλ抗HBV活性的影响,本研究以Hep G2. 2. 15细胞作为乙肝体外模型,采用脂质体转染法分别向细胞转染p EGFP-USP18、PEGFP-N1经48 h,再经IFN-α和IFN-λ处理24 h,分为阴性对照组﹑USP18过表达+IFN-α组﹑空载组+IFN-α组﹑USP18过表达+IFN-λ组﹑空载组+IFN-λ组。采用Western印迹、RT-q PCR和ELISA检测各组的乙肝病毒标志物、STAT1/p STAT1和下游的干扰素刺激基因(ISGs)的表达。结果显示,与阴性对照组和空载组相比,USP18蛋白在过表达组明显升高(P <0. 05),过表达细胞模型构建成功;在IFN-α处理的两组中,空载组中HBs Ag、HBe Ag、HBc Ag及HBV-DNA的表达均低于USP18过表达组,差异有统计学意义(P <0. 05)。而IFN-λ处理组中,乙肝病毒标志物的差异不明显。在IFN-α处理组中,空载组的ISG15、Mx A、IFIT1和p STAT1表达均高于USP18过表达组,差异有统计学意义(P <0. 05),而在IFN-λ处理组中ISGs和p STAT1的表达无明显差异。上述结果证实,USP18可通过抑制JAK/STAT信号通路的激活来减弱IFN-α抗HBV的活性。研究还证实,IFN-λ可发挥抗HBV的作用,USP18不通过JAK/STAT信号通路抑制其抗HBV活性。
Interferon alpha( IFN-α) is one of the most widely used anti-hepatitis B virus( HBV)medicine. It has been reported that ubiquitin specific protease 18( USP18) inhibits the anti-HBV activity of IFN-α,but whether USP18 has an effect on the anti-HBV activity of IFN-λ was unclear. Here we aim to detect the anti-HBV effect of USP18 on IFN-λ in an in vitro model. HepG2. 2. 15 cells were transfected with two different plasmids: the empty vector( p EGFP-N1) and USP18 overexpressed plasmid( p EGFPUSP18),and then were treated with IFN-α and IFN-λ for 24 hours,respectively. Untreated groups served as the negative control. The expression of HBV markers,STAT1/p STAT1 protein expression and interferon stimulated genes( ISGs) were tested on HepG2. 2. 15 by Western blotting,quantitative realtime PCR( RT-q PCR) and enzyme-linked immunosorbent assays( ELISA). The results showed that USP18 was successfully overexpressed in the overexpression group compared with both negative control and empty vector control groups( P < 0. 05),which demonstrated the successful establishment of vitro models. In IFN-α treated groups,the expression of hepatitis B surface antigen( HBs Ag),hepatitis B e antigen( HBe Ag),hepatitis B core antigen( HBc Ag) and HBV-DNA in overexpression groups were significantly higher than empty vectors( P < 0. 05),while there was little difference in the IFN-λtreatment group. In addition,the expression of ISG15,Mx A,IFIT1 and p STAT1 of the empty vector were obviously higher than those in overexpression groups in the treatment group of IFN-α( P < 0. 05),but there was also no significant difference in ISGs and p STAT1 expression in IFN-λ treatment groups.USP18 inhibits the anti-HBV activity of IFN-α by suppressing the activation of the JAK/STAT signaling pathway. In contrast,USP18 has no effect on the anti-HBV activity of IFN-λ via the JAK/STAT signaling pathway.
Interferon alpha( IFN-α) is one of the most widely used anti-hepatitis B virus( HBV)medicine. It has been reported that ubiquitin specific protease 18( USP18) inhibits the anti-HBV activity of IFN-α,but whether USP18 has an effect on the anti-HBV activity of IFN-λ was unclear. Here we aim to detect the anti-HBV effect of USP18 on IFN-λ in an in vitro model. HepG2. 2. 15 cells were transfected with two different plasmids: the empty vector( p EGFP-N1) and USP18 overexpressed plasmid( p EGFPUSP18),and then were treated with IFN-α and IFN-λ for 24 hours,respectively. Untreated groups served as the negative control. The expression of HBV markers,STAT1/p STAT1 protein expression and interferon stimulated genes( ISGs) were tested on HepG2. 2. 15 by Western blotting,quantitative realtime PCR( RT-q PCR) and enzyme-linked immunosorbent assays( ELISA). The results showed that USP18 was successfully overexpressed in the overexpression group compared with both negative control and empty vector control groups( P < 0. 05),which demonstrated the successful establishment of vitro models. In IFN-α treated groups,the expression of hepatitis B surface antigen( HBs Ag),hepatitis B e antigen( HBe Ag),hepatitis B core antigen( HBc Ag) and HBV-DNA in overexpression groups were significantly higher than empty vectors( P < 0. 05),while there was little difference in the IFN-λtreatment group. In addition,the expression of ISG15,Mx A,IFIT1 and p STAT1 of the empty vector were obviously higher than those in overexpression groups in the treatment group of IFN-α( P < 0. 05),but there was also no significant difference in ISGs and p STAT1 expression in IFN-λ treatment groups.USP18 inhibits the anti-HBV activity of IFN-α by suppressing the activation of the JAK/STAT signaling pathway. In contrast,USP18 has no effect on the anti-HBV activity of IFN-λ via the JAK/STAT signaling pathway.
引文
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[2] European Association for the Study of the Liver. EASL clinical practice guidelines:Management of chronic hepatitis B virus infection[J]. J Hepatol,2012,57(1):167-185
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[8] Liu LQ,Ilaria R Jr,Kingsley PD,et al. A novel ubiquitinspecific protease,UBP43,cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation[J]. Mol Cell Biol,1999,19(4):3029-3038
[9] Liu X,Li H,Zhong B,et al. USP18 inhibits NF-κB and NFAT activation during Th17 differentiation by deubiquitinating the TAK1-TAB1 complex[J]. J Exp Med, 2013, 210(8):1575-1590
[10] Katsoulidis E,Sassano A,Majchrzak-kita B,et al. Suppression of interferon(IFN)-inducible genes and IFN-mediated functional responses in BCR-ABL-expressing cells[J]. J Biol Chem,2008,283(16):10793-10803
[11] Malakhova OA,Kim KI,Luo JK,et al. UBP43 is a novel regulator of interferon signaling independent of its ISG15isopeptidase activity[J]. EMBO J,2006,25(11):2358-2367
[12] Ritchie KJ,Hahn CS,Kim KI,et al. Role of ISG15 protease UBP43(USP18)in innate immunity to viral infection[J]. Nat Med,2004,10(12):1374-1378
[13] Li L,Lei QS,Zhang SJ,et al. Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in Hep G2. 2. 15 Cells via JAK/STAT Signaling[J]. PLo S One, 2016, 11(5):e0156496
[14] Kotenko SV,Gallagher G,Baurin VV,et al. IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex[J]. Nat Immunol,2003,4(1):69-77
[15] Sheppard P,Kindsvogel W,Xu W,et al. IL-28,IL-29 and their class II cytokine receptor IL-28R[J]. Nat Immunol,2003,4(1):63-68
[16] Prokunina-Olsson L,Muchmore B,Tang W,et al. A variant upstream of IFNL3(IL28B)creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus[J]. Nat Genet,2013,45(2):164-171
[17] Lopu snáK, Re6uchováI, BetákováT, et al. Interferons lambda,new cytokines with antiviral activity[J]. Acta Virol,2013,57(2):171-179
[18] de Weerd NA,Nguyen T. The interferons and their receptors--distribution and regulation[J]. Immunol Cell Biol,2012,90(5):483-491
[19] Robek MD,Boyd BS,Chisari FV. Lambda interferon inhibits hepatitis B and C virus replication[J]. J Virol,2005,79(6):3851-3854
[20] Sells MA,Chen ML,Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA[J]. Proc Natl Acad Sci U S A,1987,84(4):1005-1009
[21] Stark GR,Damell JE Jr. The JAK-STAT pathway at twenty[J].Immunity,2012,36(4):503-514
[22] Lau GK,Piratvisuth T,Luo KX,et al. Peginterferon Alfa-2a,lamivudine,and the combination for HBe Ag-positive chronic hepatitis B[J]. N Engl J Med,2005,352(26):2682-2695
[23] Christen V,Duong F,Bernsmeier C,et al. Inhibition of alpha interferon signaling by hepatitis B virus[J]. J Virol,2007,81(1):159-165
[24] European Association for Study of Liver. EASL Clinical Practice Guidelines:management of hepatitis C virus infection[J]. J Hepatol,2014,60(2):392-420
[25] Gregorio GV, Jones H, Choudhuri K, et al. Autoantibody prevalence in chronic hepatitis B virus infection:effect in interferon alfa[J]. Hepatology,1996,24(3):520-523
[26] Malakhov MP,Malakhova OA,Kim KI,et al. UBP43(USP18)specifically removes ISG15 from conjugated proteins[J]. J Biol Chem,2002,277(12):9976-9981
[27] Chen L,Borozan I,Feld J,et al. Hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis C viral infection[J]. Gastroenterology,2005,128(5):1437-1444
[28] Yin Z,Dai J,Deng J,et al. Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells[J]. J Immunol,2012,189(6):2735-2745
[29] Zahn S,Rehkmper C,Kümmerer BM,et al. Evidence for a pathophysiological role of keratinocyte-derived type III interferon(IFNλ)in cutaneous lupus erythematosus[J]. J Invest Dermatol,2011,131(1):133-140
[30] Sommereyns C,Paul S,Staeheli P,et al. IFN-lambda(IFNlambda)is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo[J]. PLo S Pathog,2008,4(3):e1000017
[31] Kohli A,Zhang X,Yang J,et al. Distinct and overlapping genomic profiles and antiviral effects of Interferon-λand-αon HCV-infected and noninfected hepatoma cells[J]. J Viral Hepat,2012,19(12):843-853
[32] Hou W,Wang X,Ye L,et al. Lambda interferon inhibits human immunodeficiency virus type 1 infection of macrophages[J]. J Virol,2009,83(8):3834-3842
[33] Arnold MM,Sen A,Greenberg HB,et al. The battle between rotavirus and its host for control of the interferon signaling pathway[J]. PLo S Pathog,2013,9(1):e1003064
[34] Coccia EM,Severa M,Giacomini E,et al. Viral infection and Toll-like receptor agonists induce a differential expression of type I and lambda interferons in human plasmacytoid and monocytederived dendritic cells[J]. Eur J Immunol,2004,34(3):796-805
[35] Doyle SE,Schreckhise H,Khuu-Duong K,et al. Interleukin-29uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes[J]. Hepatology,2006,44(4):896-906
[36] Friborg J,Levine S,Chen C,et al. Combinations of lambda interferon with direct-acting antiviral agents are highly efficient in suppressing hepatitis C virus replication[J]. Antimicrob Agents Chemother,2013,57(3):1312-1322
[2] European Association for the Study of the Liver. EASL clinical practice guidelines:Management of chronic hepatitis B virus infection[J]. J Hepatol,2012,57(1):167-185
[3] Orlando R,Foggia M,Maraolo AE,et al. Prevention of hepatitis B virus infection:from the past to the future[J]. Eur J Clin Microbl Infect Dis,2015,34(6):1059-1070
[4] Xiao C,Qin B,Chen L,et al. Preactivation of the interferon signalling in liver is correlated with nonresponse to interferon alpha therapy in patients chronically infected with hepatitis B virus[J]. J Viral Hepat,2012,19(2):e1-10
[5] Wang J,Jiang D,Zhang H,et al. Proteome responses to stable hepatitis B virus transfection and following interferon alpha treatment in human liver cell line Hep G2[J]. Proteomics,2009,9(6):1672-1682
[6] Chen H,Wang LW,Huang YQ,et al. Interferon-alpha induces high expression of APOBEC3G and STAT-1 in vitro and in vivo[J]. Int J Mol Sci,2010,11(9):3501-3512
[7] Wieland SF,Vega RG,Müller R,et al. Searching for interferoninduced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes[J]. J Virol,2003,77(2):1227-1236
[8] Liu LQ,Ilaria R Jr,Kingsley PD,et al. A novel ubiquitinspecific protease,UBP43,cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation[J]. Mol Cell Biol,1999,19(4):3029-3038
[9] Liu X,Li H,Zhong B,et al. USP18 inhibits NF-κB and NFAT activation during Th17 differentiation by deubiquitinating the TAK1-TAB1 complex[J]. J Exp Med, 2013, 210(8):1575-1590
[10] Katsoulidis E,Sassano A,Majchrzak-kita B,et al. Suppression of interferon(IFN)-inducible genes and IFN-mediated functional responses in BCR-ABL-expressing cells[J]. J Biol Chem,2008,283(16):10793-10803
[11] Malakhova OA,Kim KI,Luo JK,et al. UBP43 is a novel regulator of interferon signaling independent of its ISG15isopeptidase activity[J]. EMBO J,2006,25(11):2358-2367
[12] Ritchie KJ,Hahn CS,Kim KI,et al. Role of ISG15 protease UBP43(USP18)in innate immunity to viral infection[J]. Nat Med,2004,10(12):1374-1378
[13] Li L,Lei QS,Zhang SJ,et al. Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in Hep G2. 2. 15 Cells via JAK/STAT Signaling[J]. PLo S One, 2016, 11(5):e0156496
[14] Kotenko SV,Gallagher G,Baurin VV,et al. IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex[J]. Nat Immunol,2003,4(1):69-77
[15] Sheppard P,Kindsvogel W,Xu W,et al. IL-28,IL-29 and their class II cytokine receptor IL-28R[J]. Nat Immunol,2003,4(1):63-68
[16] Prokunina-Olsson L,Muchmore B,Tang W,et al. A variant upstream of IFNL3(IL28B)creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus[J]. Nat Genet,2013,45(2):164-171
[17] Lopu snáK, Re6uchováI, BetákováT, et al. Interferons lambda,new cytokines with antiviral activity[J]. Acta Virol,2013,57(2):171-179
[18] de Weerd NA,Nguyen T. The interferons and their receptors--distribution and regulation[J]. Immunol Cell Biol,2012,90(5):483-491
[19] Robek MD,Boyd BS,Chisari FV. Lambda interferon inhibits hepatitis B and C virus replication[J]. J Virol,2005,79(6):3851-3854
[20] Sells MA,Chen ML,Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA[J]. Proc Natl Acad Sci U S A,1987,84(4):1005-1009
[21] Stark GR,Damell JE Jr. The JAK-STAT pathway at twenty[J].Immunity,2012,36(4):503-514
[22] Lau GK,Piratvisuth T,Luo KX,et al. Peginterferon Alfa-2a,lamivudine,and the combination for HBe Ag-positive chronic hepatitis B[J]. N Engl J Med,2005,352(26):2682-2695
[23] Christen V,Duong F,Bernsmeier C,et al. Inhibition of alpha interferon signaling by hepatitis B virus[J]. J Virol,2007,81(1):159-165
[24] European Association for Study of Liver. EASL Clinical Practice Guidelines:management of hepatitis C virus infection[J]. J Hepatol,2014,60(2):392-420
[25] Gregorio GV, Jones H, Choudhuri K, et al. Autoantibody prevalence in chronic hepatitis B virus infection:effect in interferon alfa[J]. Hepatology,1996,24(3):520-523
[26] Malakhov MP,Malakhova OA,Kim KI,et al. UBP43(USP18)specifically removes ISG15 from conjugated proteins[J]. J Biol Chem,2002,277(12):9976-9981
[27] Chen L,Borozan I,Feld J,et al. Hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis C viral infection[J]. Gastroenterology,2005,128(5):1437-1444
[28] Yin Z,Dai J,Deng J,et al. Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells[J]. J Immunol,2012,189(6):2735-2745
[29] Zahn S,Rehkmper C,Kümmerer BM,et al. Evidence for a pathophysiological role of keratinocyte-derived type III interferon(IFNλ)in cutaneous lupus erythematosus[J]. J Invest Dermatol,2011,131(1):133-140
[30] Sommereyns C,Paul S,Staeheli P,et al. IFN-lambda(IFNlambda)is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo[J]. PLo S Pathog,2008,4(3):e1000017
[31] Kohli A,Zhang X,Yang J,et al. Distinct and overlapping genomic profiles and antiviral effects of Interferon-λand-αon HCV-infected and noninfected hepatoma cells[J]. J Viral Hepat,2012,19(12):843-853
[32] Hou W,Wang X,Ye L,et al. Lambda interferon inhibits human immunodeficiency virus type 1 infection of macrophages[J]. J Virol,2009,83(8):3834-3842
[33] Arnold MM,Sen A,Greenberg HB,et al. The battle between rotavirus and its host for control of the interferon signaling pathway[J]. PLo S Pathog,2013,9(1):e1003064
[34] Coccia EM,Severa M,Giacomini E,et al. Viral infection and Toll-like receptor agonists induce a differential expression of type I and lambda interferons in human plasmacytoid and monocytederived dendritic cells[J]. Eur J Immunol,2004,34(3):796-805
[35] Doyle SE,Schreckhise H,Khuu-Duong K,et al. Interleukin-29uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes[J]. Hepatology,2006,44(4):896-906
[36] Friborg J,Levine S,Chen C,et al. Combinations of lambda interferon with direct-acting antiviral agents are highly efficient in suppressing hepatitis C virus replication[J]. Antimicrob Agents Chemother,2013,57(3):1312-1322