USP4通过去泛素化RIG-I正向调节其介导的抗病毒作用
摘要
Ⅰ型干扰素(IFN-α/β)具有较强的抗病毒活性,并在机体对抗病毒感染中发挥举足轻重的作用。病毒感染机体后,其保守的病原相关分子模式可被Toll样受体(Toll like receptors)、维甲酸诱导基因Ⅰ受体RIG-Ⅰ-like receptors, RLRs)等模式识别受体(Pattern-recognition receptors, PRRs)所识别,通过一系列信号转导,最终激活免疫细胞释放Ⅰ型干扰素及促炎症细胞因子,从而发挥抗病毒作用。
蛋白的泛素化作用在RIG-Ⅰ受体的活化和抗病毒免疫反应中起着至关重要的调节作用。然而,与RIG-Ⅰ活化相关的去泛素化作用的功能尚不明确。我们研究发现,泛素特异性蛋白酶(Ubiquitin-specific protease4, USP4)可以通过其去泛素化酶活性来调节RIG-Ⅰ的表达和活化。病毒感染诱导RIG-Ⅰ激活后,USP4的表达明显降低。而USP4的过表达能够显著增强RIG-Ⅰ的表达并上调其活化诱导的IFN-β的释放,同时抑制水泡口炎病毒(Vesicular stomatitis virus, VSV)的复制增殖。而用siRNA干扰USP4表达后会得到相反的结果。另外,USP4可以与RIG-Ⅰ直接相互作用并特异性去除其K48偶联的泛素链。
因此,我们提出,USP4作为RIG-Ⅰ的一个新的调节分子,通过其去泛素化酶活性去除RIG-Ⅰ K48偶联的泛素链来稳定RIG-Ⅰ的表达,从而正向调节其下游信号通路。
研究目的：
1、闸明USP4在RIG-Ⅰ信号通路中的功能；
2、找出USP4的作用靶点及机制；
3、探讨USP4在细胞抗病毒反应中的作用。
研究方法：
1、在不同细胞中检测RIG-Ⅰ的活化对USP4的表达的影响。
1.1、在Hela细胞中检测RIG-Ⅰ活化对USP4表达的影响。
a.Ⅰ在Hela细胞中转染poly(I:C)0,2,4,8,12h,RT-PCR检测USP4的表达(mRNA水平)；Ⅱ在Hela细胞中转染poly(I:C)0,12,24,36h,Western blot检测USP4的表达(蛋白水平)。
b.Ⅰ用SeV(Sendai virus,仙台病毒)刺激Hela细胞0,8,12,24h,RT-PCR检测USP4的表达(mRNA水平)；Ⅱ用SeV刺激Hela细胞0,8,12,24,36h,Western blot检测USP4的表达(蛋白水平)。
1.2在小鼠原代腹腔巨噬细胞中检测RIG-Ⅰ活化对USP4表达的影响。提取小鼠原代腹腔巨噬细胞,
ⅠSeV刺激0,4,8,12,24h,RT-PCR检测USP4的表达(mRNA水平)；
ⅡSeV刺激0,12,24,36h,Western blot检测USP4的表达(蛋白水平)。
2、表达变化的USP4对RIG-Ⅰ介导的IFN-β表达的影响。
2.1mRNA水平检测表达变化的USP4对RIG-Ⅰ介导的IFN-β表达的影响。
a.在HEK293细胞中分别转染空白质粒或USP4的高表达质粒24h后,再分别转染poly(I:C)或poly(dA:dT)12h,RT-PCR检测IFN-β的表达。
b.Ⅰ在HEK293或Hela细胞中分别转染Ctrl siRN八或USI'4siRNA36h后,再转染poly(I:C)0,4h,811,RT-PCR检测IFN-β的表达。Ⅱ在HEK293或Hela细胞中分别转染Ctrl siRN八或USP4siRNA36h后,再转染poly(dA:dT)0,16h,RT-PCR检测IFN-β的表达。Ⅲ在Hela细胞中分别转染Ctrl siRN八或USP4siRNA36h后,SeV刺激0,12h,RT-PCR检测IFN-β的表达。
2.2检测表达变化的USP4对RIG-Ⅰ介导的IFN-β和IRF3报告基因活性的影响。
a.在Hela细胞中分别共转染空白质粒或USP4的高表达质粒和IFN-β、IRF3的报告基因质粒,SeV刺激12h后检测报告基因活性差异。
b.在Hela细胞中分别转染Ctrl siRNA或USP4siRNA,再分别转染IFN-β、 IRF3的报告基因质粒,SeV刺激12h后检测报告基因活性差异。
3、确定USP4的作用靶点。
3.1mRNA水平检测表达变化的USP4对RIG-Ⅰ信号通路中各接头分子介导的IFN-β表达的影响。
a.在HEK293细胞中分别共转染空白质粒或USP4的高表达质粒和RIG-Ⅰ， MAVS, TBK1, IRF35D, TRIF, IKK-ε, MDA5的表达质粒24h, RT-PCR检测IFN-β的表达。
b.在HEK293细胞中分别转染Ctrl siRNA或USP4siRNA24h后,再分别转染RIG-Ⅰ, MAVS, TBK1, IRF35D, MDA5的表达质粒24h, RT-PCR检测IFN-β的表达。
c.在Huh7.5细胞中分别共转染空白质粒或USP4的高表达质粒和RIG-I,MAVS,TBK1,IRF35D的表达质粒24h,RT-PCR检测IFN-β的表达。
3.2检测USP4高表达对RIG-I信号通路中各接头分子介导的IFN-β和IRF3报告基因活性的影响。
在HEK293细胞中分别共转染空白质粒或USP4的高表达质粒和TRIF, RIG-Ⅰ, MAVS, TBK1, IRF35D的表达质粒与IFN-β、IRF3的报告基因质粒,24h后检测报告基因活性差异。
3.3蛋白水平检测表达变化的USP4对各接头分子表达的影响。
a.在HEK293细胞中分别共转染不同剂量的空白质粒或USP4的高表达质粒和RIG-Ⅰ, MAVS, STING TBK1, IRF3, IKK-ε, MDA5的表达质粒,Western Blot检测各接头分子的表达情况。
b.在Hela细胞中分别转染Ctrl siRNA或USP4siRNA36h, Western Blot检测内源性的RIG-Ⅰ, MAVS和TBK1的表达情况。
4、USP4调控RIG-I信号通路的分子机制研究。
4.1根据前面的结果,免疫共沉淀明确USP4与RIG-Ⅰ、MAVS、TBK1等相关靶分子的结合情况。
a.在HEK293细胞中分别共转染带有Myc标签的USP4质粒和带有Flag标签的RIG-Ⅰ、MAVS等质粒,分别用相应抗体进行免疫沉淀检测其相互结合情况。
b.在Hela细胞中转染poly(I:C)0,8,12h,免疫沉淀结合Western Blot检测内源性的USP4与RIG-Ⅰ、MAVS等信号分子的结合情况。
c.在HEK293细胞中分别共转染USP4各节段质粒和RIG-Ⅰ、MAVS等质粒,免疫共沉淀检测各节段与靶分子的结合情况,明确USP4与靶分子结合的结构域。
4.2USP4对相应靶分子泛素化的影响。
a.靶分子表达质粒、USP4表达质粒或空白质粒分别与泛素质粒共转染HEK293细胞,免疫共沉淀结合Western Blot检测USP4对靶分子泛素化的影响。
b.靶分子表达质粒、USP4表达质粒或空白质粒分别与泛素质粒野生型或突变体(K48突变体、K63突变体)共转染HEK293细胞,免疫共沉淀结合Western Blot检测USP4对靶分子泛素化形成(K48或K63)的影响。
c.在HEK293细胞中分别共转染Ctrl siRNA或USP4siRNA后,再共转染靶分子表达质粒和泛素质粒野生型或突变体(K48突变体、K63突变体),免疫共沉淀结合Western Blot检测USP4对靶分子泛素化形式(K48或K63)的影响。
d.在Hela细胞中分别转染Ctrl siRNA或USP4siRNA, SeV刺激4h后,免疫共沉淀结合Western Blot检测USP4对内源性靶分子泛素化的影响,同时明确泛素化形式(K48或K63)。
5、USP4在抗病毒感染中的功能研究。
a.在HEK293细胞中分别转染空白质粒或USP4的高表达质粒24h后,用VSV感染细胞,检测VSV的复制情况。(空斑形成实验检测病毒滴度；实时定量PCR检测细胞中VSV mRNA)
b.在HEK293细胞中分别共转染Ctrl siRNA或USP4siRNA48h后,用VSV感染细胞,检测VSV的复制情况。(空斑形成实验检测病毒滴度,实时定量PCR检测细胞中VSV mRNA)
研究结果：
1、RIG-Ⅰ信号通路的活化能够抑制USP4的表达。
a.用poly(I:C)转染或SeV刺激Hela细胞以激活RIG-Ⅰ信号通路后,USP4的表达被明显抑制(mRNA水平和蛋白水平)。
b.用SeV刺激小鼠原代腹腔巨噬细胞,USP4的表达也明显降低(mRNA水平和蛋白水平)。
2、USP4可以显著增强RIG-Ⅰ介导的IFN-β的表达。
a.在HEK293细胞中转染USP4的高表达质粒后,转染poly(I:C)或poly(dA：dT)(活化RIG-Ⅰ信号通路)诱导的IFN-β表达明显上调。
b.特异性小干扰RNA抑制HEK293细胞或Hela细胞中内源性USP4的表达,转染poly(I:C)或poly(dA:dT)诱导的IFN-β表达被明显抑制。
c.特异性小干扰RNA抑制Hela细胞中内源性USP4的表达,可显著抑制SeV诱导的IFN-β的表达。
d.在Hela细胞中,USP4的高表达可明显增强SeV介导的IFN-β和IRF3报告基因的活化；特异性小干扰RNA抑制USP4的表达后,SeV介导的IFN-β和IRF3报告基因活化被显著抑制。
3、USP4靶向作用于RIG-Ⅰ。
3.1USP4特异性调控RIG-Ⅰ介导的IFN-β表达(mRNA水平)。
a.USP4的过表达可明显增强HEK293细胞中RIG-Ⅰ介导的IFN-β表达(mRNA水平),而对其下游信号分子MAVS, STING, TBK1和接头蛋白MDA5介导的IFN-β的表达无明显影响。在RIG-Ⅰ缺陷的Huh7.5细胞中结果类似。
b.特异性小干扰RNA抑制HEK293细胞中内源性USP4的表达,可显著抑制RIG-Ⅰ介导的IFN-β表达(mRNA水平),但对其下游信号分子MAVS, STING, TBK1, IRF3, IKK-ε和接头蛋白MDA5介导的IFN-β的表达作用不大。
3.2USP4特异性调控RIG-Ⅰ介导IFN-β及IRF3的报告基因活化。
USP4的过表达可明显增强HEK293细胞中RIG-Ⅰ介导的IFN-β及IRF3的报告基因活化,而对其下游信号分子MAVS，TBK1，IRF3及接头分子TRIF介导的IFN-β及IRF3的报告基因活化无明显作用。
3.3USP4特异性增强RIG-Ⅰ的表达。
a.在HEK293细胞中,USP4的过表达能够显著上调外源性RIG-Ⅰ的表达,且具有剂量依赖性；而对于外源性MAVS,STING、TBK1,IRF3,IKK-ε, MDA5的表达无明显影响。
b.特异性小干扰RNA抑制Hela细胞中内源性USP4的表达,可显著并抑制内源性RIG-Ⅰ的表达,而对内源州MAVS.TBK1的表达无明显作用。
4.USP4可以特异性与RIG-Ⅰ相结合。
4.1USP4可以与RIG-Ⅰ相互结合。
a.在HEK293细胞中共转染带有不同标签的USP4和RIG-Ⅰ的表达质粒,分别用相应抗体进行免疫共沉淀,Western Blot的结果显示,外源性的USP4和RIG-Ⅰ可以相互结合。
b.在Hela细胞中，转染poly(I:C)不同的时间点后,用内源性的抗体进行免疫共沉淀,Western Blot的结果显示,内源性的USP4和RIG-Ⅰ能够组成性结合。
4.2USP4特异性结合于RIG-Ⅰ的N末端Card结构域。
在HEK293细胞中分别共转染空白质粒或USP4表达质粒与RIG-Ⅰ仅有N末端两个Card结构域的质粒RIG-IN,Western Blot的结果显示,USP4的过表达可以显著增强RIG-IN的表达；免疫共沉淀结果表明,USP4可以直接与RIG-IN相结合。
4.3USP4与RIG-Ⅰ的结合位点位于其N末端的DUSP结构域。
在HEK293细胞中分别共转染USP4的不同节段质粒(WT，野生型；N260,C末端USP结构域缺失：C261,N末端DUSP结构域缺失)与RIG-Ⅰ表达质粒,免疫共沉淀结果表明,RIG-Ⅰ与WT,N260可以相结合,但是与C261末检测到结合。
5.USP4可以特异性移除RIG-Ⅰ的K48-偶联的泛素链。
5.1USP4的过表达可特异性下调RIG-ⅠK48-偶联的体外泛素化作用。
a. RIG-Ⅰ表达质粒、USP4表达质粒或空白质粒分别与泛素粒共转染HEK293细胞,免疫共沉淀结果表明,USP4过表达能明显下调RIG-Ⅰ的体外泛素化作用。
b. RIG-Ⅰ表达质粒、USP4表达质粒或空白质粒分别与泛素质粒野生型或突变体(K48突变体、K63突变体)共转染HEK293细胞,免疫共沉淀结果显示,USP4过表达可特异性下调RIG-Ⅰ的K48偶联的泛素化。同时,USP4对于MAVS的体外泛素化作用无明显影响。
5.2特异性小干扰RNA抑制HEK293细胞中内源性USP4的表达,可显著上调RIG-ⅠK48-偶联的体外泛素化作用。
a.在HEK293细胞中分别共转染Ctrl siRNA或USP4siRNA后,再共转染RIG-I表达质粒和泛素质粒野生型或突变体(K48突变体、K63突变体),免疫共沉淀结果显示,USP4的低表达能特异性增强RIG-Ⅰ K48-偶联的体外泛素化作用。
b.小干扰RNA抑制Hela细胞中内源性USP4的表达,可显著上调SeV刺激诱导的RIG-Ⅰ内源性泛素化作用,同时其K48-偶联的泛素化作用也明显增强。
6.USP4参与细胞抗病毒反应。USP4的过表达能明显抑制VSV病毒在HEK293细胞中的复制；而用小干扰RNA抑制内源性USP4的表达后能显著促进VSV病毒在胞内的复制。
结论：
1、RIG-Ⅰ信号通路的活化能够抑制USP4的表达。
2、USP4能特异性增强RIG-Ⅰ介导的IFN-β的表达。
3、USP4可以与RIG-β相结合并通过去泛素化酶活性去除其K48-偶联的泛素链来稳定RIG-β的表达,从而正向调节其下游信号通路。
4、USP4参与细胞抗病毒反应。
创新点及意义：
1、本研究首次提出,USP4作为RIG-Ⅰ的一个新的调节分子,通过其去泛素化酶活性去除RIG-Ⅰ K48偶联的泛素链来稳定RIG-Ⅰ的表达,从而正向调节其下游信号通路。
2、本研究为正向调控RLR信号通路提供新的实验证据，为抗病毒研究、诊断和治疗提供新的思路。
Type I IFNs (IFN-a/(3) possess strong anti-viral activity and play pivotal roles in defense against viral infection. During viral infection, pattern-recognition receptors (PRRs), including TLRs and retinoic acid-inducible gene-Ⅰ (RIG-I)-like helicases (RLRs), activate immune cells to produce type IIFN and proinflammatory cytokines, which are involved in the elimination of virus.
Protein ubiquitination plays an essential role in the regulation of RIG-I activation and antiviral immune response. However, the function of the opposite process deubiquitination in RIG-Ⅰ activation remains elusive. In this study, we have identified the deubiquitinating enzyme Ubiquitin-specific protease4(USP4) as a new regulator for RIG-I activation through deubiquitination and stabilization of RIG-Ⅰ. USP4expression was attenuated after virus-induced RIG-Ⅰ activation. Overexpression of USP4significantly enhanced RIG-Ⅰ protein expression and RIG-Ⅰ-triggered IFN-β signaling, at the same time, inhibited VSV replication. siRNA knockdown of USP4expression has an opposite effect. Furthermore, USP4was found to interact with RIG-Ⅰ and remove K48-linked polyubiquitinattion chains from RIG-Ⅰ.
Therefore, our study identified USP4as a new positive regulator for RIG-Ⅰ through deubiquitinating K48-linked ubiquitin chains and stabilizing RIG-Ⅰ.
Objectives:
1. To investigate the function of USP4in RIG-I signaling;
2. To determine the molecular mechanisms and targets of USP4;
3. To investigate the effect of USP4on antiviral response.
Methods:
1. Identification differentially expressed USP4upon RIG-I activation in different cell lines.
1.1Identification differentially expressed USP4upon RIG-I activation in Hela cells.
a. I RT-PCR analysis of USP4expression in Mela cells transfected with poly(I:C) for0,2,4,8,12h (mRNA level);
II Western blot analysis of USP4expression in Mela cells transfected with poly(I:C) for0,12,24,36h (protein level).
b. I RT-PCR analysis of USP4expression in Hela cells infected with SeV (Sendai virus) for0,8,12,24h (mRNA level);
II Western blot analysis of USP4expression in Mela cells infected with SeV for0,8,12,24,36h (protein level).
1.2Identification differentially expressed USP4upon RIG-I activation in mouse peritoneal macrophages.
After bone marrow derived macrophages (BMDM) were infected with SeV for0,4,8,12,24h, RT-PCR analysis of USP4expression (mRNA level); infected with SeV for0,12,24,36h. Western blot analysis of USP4expression (protein level).
2. Confirmation of whether the RIG-I-induced IFN-β production was regulated by USP4.
2.1RT-PCR analysis whether the RIG-I-induced IFN-β production was regulated by USP4on mRNA level.
a. HEK293cells transfected with the indicated plasmids were transfected with poly(I:C) or poly(dA:dT) for12h. Total RNA was prepared and analyzed for the expressions of IFN-β, USP4and GAPDH by RT-PCR.
b.ⅠHEK293or Hela cells were transfected with control siRNA or USP4siRNA for36h, and then transfected with poly(I:C) or for0,4h,8h, Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.
Ⅱ HEK293or Hela cells were transfected with control siRNA or USP4siRNA for36h, and then transfected with poly(dA:dT) or for0,16h, Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.
Ⅲ Hela cells were transfected with control siRNA or USP4siRNA for36h, and then infected with SeV for12h. Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.
2.2Luciferase assay analysis whether the RIG-I-induced IFN-P promoter and IRF3activation was regulated by USP4.
a. Hela cells were transiently transfected with IFN-β or IRF3reporter plasmid together with USP4expression plasmid or control plasmid, analyzed luciferase activity after SeV infection for12h.
b. Hela cells were transfected with control siRNA or USP4siRNA, and then transfected with IFN-P or IRF3reporter plasmid, analyzed luciferase activity after SeV infection for12h.
3、To determine the molecular targets of USP4.
3.1RT-PCR analysis the IFN-β production induced by RIG-I or adaptors in RIG-I pathway on mRNA level modulated upon differentially expression of USP4.
a. HEK293cells were transfected with RIG-I, MAVS, TBK1, IRF35D, TRIF, IKK-e, MDA5along with USP4plasmid, and24h later IFN-P mRNA were analyzed by RT-PCR.
b. HEK293cells were transfected with RIG-I, MAVS, TBK1, IRF35D, MDA5along with USP4specific siRNA or control siRNA, and IFN-P mRNA were analyzed by RT-PCR. c. Huh7.5cells were transfected with RIG-I, MAVS, IRF35D along with USP4plasmid, and24h later IFN-β mRNA were analyzed by RT-PCR.
3.2Luciferase assay analysis the RIG-I or other adaptors in RIG-I pathway induced IFN-β promoter and IRF3activation was regulated upon overexpression of USP4.
HEK293cells were transfected with TRIF, RIG-I, MAVS, TBK1, IRF35D along with IFN-β or IRF3reporter plasmid and USP4plasmid, and24h later luciferase activity was analyzed.
3.3Western blot analysis of expression of adaptors in RIG-I pathway upon differentially expression of USP4.
a. Western blot analysis of the lysates from HEK293cells transfected with Myc-RIG-I, MAVS, STING, TBK1, IRF3, IKK-ε, MDA5together with increasing concentration of Flag-USP4expression plasmid.
b. Western blot analysis of the expression of RIG-I, MAVS, and TBK1in Hela cells transfected with control siRNA or USP4siRNA and36h later infected with SeV for indicated time periods.
4. To clarify the molecular mechanisms of USP4.
4.1To investigate the interaction between USP4and the target molecules by immunopreicpitation.
a. Lysates from HEK293cells transiently cotransfected with Flag-tag target molecules and Myc-USP4expression plasmids were subjected to immunoprecipitation with anti-Myc or anti-Flag antibody followed by western blot analysis with anti-Flag or anti-Myc antibody.
b. Hela cells transfected with poly(I:C) for0,8,12h were subjected to immunoprecipitation with anti-USP4or control IgG followed by western blot analysis with specific antibody. Proteins in whole-cell lysate were used as positive control (Input).
c. Lysates from HEK293cells transiently cotransfected with Flag-tagged target molecule and HA-tagged USP4mutants were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.
4.2To test the role of USP4in ubiquitination of the target molecular.
a. Lysates from HEK293cells transiently cotransfected with Flag-target molecule, Myc-USP4and HA-Ub plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.
b. Lysates from HEK293cells transiently cotransfected with Flag-target molecule, Myc-USP4or vector control and HA-Ub (WT), HA-Ub (K48) or HA-Ub (K63) plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.
c. Lysates from HEK293cells transfected with control siRNA or USP4siRNA and Flag-target molecule and HA-Ub (WT), HA-Ub (K48) or HA-Ub (K63) plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.
d. Lysates from Hela cells transfected with control siRNA or USP4siRNA and infected with SeV for4h were subjected to immunoprecipitation with specific antibody followed by western blot analysis with anti-Ub or anti-Ub(K48) antibody.5. To investigate the effect of USP4on antiviral response.
a. HEK293cells (2×105) transfected with the indicated plasmids (1μ g each).24h later, cells were further transfected with poly(I:C)(0.1μ g) or left untreated.18h after poly(I:C) transfection, cells were infected with VSV (MO1,0.1), and the supernatants were harvested at12h post-infection. Supernatants were analyzed for VSV titers with standard plaque assays. Intracellular VSV RNA replicates were measured by QRT-PCR.
b. HEK293cells (2×105) were transfected with control siRNA or USP4siRNA and then treated as in a.
Results:
1. USP4expression is inhibited by RIG-Ⅰ activation.
a. Transfection of poly(I:C) or infection of SeV, which has been shown to activate RIG-I signaling substantially attenuated USP4mRNA and protein expression in Hela cells.
b. SeV infection could also attenuate USP4expression in murine peritoneal macrophages.
2. USP4enhances RIG-Ⅰ-induced IFN-β production.
a. In HEK293cells, overexpression of USP4further increased poly(I:C)-induced expression of IFN-β.
b. USP4knockdown through siRNA transfection significantly inhibited poly(I:C) or poly(dA:dT)-induced IFN-β expression in HEK293cells. Similarly, poly(I:C) or poly(dA:dT)-induced IFN-β production was also greatly attenuated by USP4knockdown in Hela cells.
c. SeV-induced IFN-β production was substantially attenuated after knockdown of USP4expression in Hela cells.
d. USP4overexpression significantly enhanced SeV-induced IFN-β promoter and IRF3activation in Hela cells. In contrast, USP4knockdown significantly inhibited SeV-induced IFN-β promoter and IRF3activation.
3. USP4targets RIG-I.
3.1USP4specifically enhanced RIG-Ⅰ-induced IFN-β mRNA expression.
a. In HEK293cells, USP4enhanced RIG-Ⅰ-induced IFN-β mRNA expression, while, MDA5-, MAVS-, TBK1-, IKK-ε-, TRIF-and IRF35D-induced IFN-β mRNA expression was not impaired by USP4overexpression. Similarly, RIG-Ⅰ-induced IFN-β mRNA expression was also attenuated by USP4overexpression in Huh7.5cells.
b. In HEK293cells, USP4siRNA transfection substantially decreased RIG-induced IFN-β mRNA expression, but not MDA5-, MAVS-, TBK1-, IKK-ε-, TRIF and IRF35D-induced IFN-β mRNA expression.
3.2USP4specifically enhanced RIG-Ⅰ-induced activation of IFN-β promoter and IRF3reporter.
RIG-Ⅰ-induced activation of IFN-β promoter and IRF3reporter were also significantly enhanced by USP4overexpression. But, MAVS-, TBK1, TRIF and IRF3-induced activation of IFN-β promoter and IRF3reporter was not affected by USP4ovexpression.
3.3USP4specifically enhanced RIG-Ⅰ expression.
a. USP4expression significantly enhanced RIG-Ⅰ expression in a dose dependent manner. In contrast, USP4overexpression had no effects on MDA5, MAVS, STING, TBK1, IKK-ε and IRF3expression.
b. Knockdown of USP4expression significantly decreased RIG-Ⅰ protein expression especially. As a control, MAVS and TBK1protein levels were not impaired.
4. USP4specifically interacts with RIG-Ⅰ.
4.1USP4interacts with RIG-Ⅰ.
a. Myc-USP4and Flag-RIG-I were cotransfected into HEK293cells,24h after transfection, immunoprecipitation experiments were performed with Myc or Flag antibodies. Flag-RIG-I was coprecipitated with Myc-USP4using anti-Myc antibody and vice versa.
b. Endogenous interaction between RIG-Ⅰ and USP4was examined in Hela cells transfected with poly(I:C) for various times by immunopreicpitation with USP4antibody and western blotting with RIG-Ⅰ antibody. USP4was found constitutively to interact with RIG-Ⅰ. As a control, the interaction could not be detected with normal IgG.
4.2USP4interacts with the N-terminal domain of RIG-Ⅰ.
Two RIG-Ⅰ truncations (N-terminal domain composed of two CARD motifs and a C-terminal domain) were used in the immunoprecipitation assays. USP4was found to interact with the N-terminal domain of RIG-Ⅰ. Consistently, overexpression of USP4could also increase the protein expression of the RIG-Ⅰ mutant RIG-Ⅰ-N harboring the N-terminal domain.
4.3USP4and RIG-Ⅰ form a complex through the DUSP domain of USP4and the N-terminal domain of RIG-Ⅰ.
Coimmunoprecipitation experiments showed that RIG-Ⅰ interacted with WT USP4and the N-terminal DUSP domain, but not the C-terminal USP domain.
5. USP4removes K48-linked polyubiquitination conjugate from RIG-I.
5.1USP4overexpression remarkably decreased RIG-Ⅰ K48-linked ubiquitination.
a. RIG-Ⅰ was co-expressed with HA-ubiquitin and USP4or vector control. Cotransfection of USP4expression plasmid remarkably decreased RIG-Ⅰ ubiquitination.
b. Ubiquitin mutant vectors K48and K63, which contain arginine substitutions of all of its lysine residues except the one at position48and63respectively, were used in the transfection assays. Cotransfection of USP4greatly decreased the polyubiqutination of RIG-Ⅰ in the setting of ubiquitin WT and K48transfected cells, but not in the K63-transfected cells. In contrast, neither K48-linked nor K63-linked ubiquitination of MAVS was impaired by USP4overexpression.
5.2K-48-linked polyubiqutination of RIG-I was greatly increased after USP4knockdown.
a. The level of RIG-Ⅰ polyubiqutination was greatly increased after transfection of USP4siRNA in the setting of WT ubiquitin and ubiquitin mutant K.48transfected cells compared to control siRNA transfected cells, but not in mutant K63transfected cells.
b. Transfection of USP4siRNA further increased K48-linked polyubiqutination of RIG-Ⅰ in SeV-transfected Mela cells.
6. USP4is involved in cellular antiviral response.
Plaque assays of HEK.293cells infected with VSV showed that overexpression of USP4substantially decreased viral replication compared to control vector-transfected cells. And transfection of USP4siRNA greatly increased VSV viral replication in HEK.293cells.
Conclusion:
1、USP4expression is inhibited by RIG-I activation.
2、USP4enhances RIG-I-induced IFN-β production.
3、USP4positively regulates RIG-I pathway by interacting with RIG-I and removing K48-linked polyubiquitinattion chains from RIG-I.
4、USP4is involved in cellular antiviral response.
Innovation and significances:
1. We identified USP4as a new positive regulator for RIG-I through deubiquitinating K48-linked ubiquitin chains and stabilizing RIG-I.
2. Our study provided new experimental evidence for negative regulation anti-virus research.
蛋白的泛素化作用在RIG-Ⅰ受体的活化和抗病毒免疫反应中起着至关重要的调节作用。然而,与RIG-Ⅰ活化相关的去泛素化作用的功能尚不明确。我们研究发现,泛素特异性蛋白酶(Ubiquitin-specific protease4, USP4)可以通过其去泛素化酶活性来调节RIG-Ⅰ的表达和活化。病毒感染诱导RIG-Ⅰ激活后,USP4的表达明显降低。而USP4的过表达能够显著增强RIG-Ⅰ的表达并上调其活化诱导的IFN-β的释放,同时抑制水泡口炎病毒(Vesicular stomatitis virus, VSV)的复制增殖。而用siRNA干扰USP4表达后会得到相反的结果。另外,USP4可以与RIG-Ⅰ直接相互作用并特异性去除其K48偶联的泛素链。
因此,我们提出,USP4作为RIG-Ⅰ的一个新的调节分子,通过其去泛素化酶活性去除RIG-Ⅰ K48偶联的泛素链来稳定RIG-Ⅰ的表达,从而正向调节其下游信号通路。
研究目的：
1、闸明USP4在RIG-Ⅰ信号通路中的功能；
2、找出USP4的作用靶点及机制；
3、探讨USP4在细胞抗病毒反应中的作用。
研究方法：
1、在不同细胞中检测RIG-Ⅰ的活化对USP4的表达的影响。
1.1、在Hela细胞中检测RIG-Ⅰ活化对USP4表达的影响。
a.Ⅰ在Hela细胞中转染poly(I:C)0,2,4,8,12h,RT-PCR检测USP4的表达(mRNA水平)；Ⅱ在Hela细胞中转染poly(I:C)0,12,24,36h,Western blot检测USP4的表达(蛋白水平)。
b.Ⅰ用SeV(Sendai virus,仙台病毒)刺激Hela细胞0,8,12,24h,RT-PCR检测USP4的表达(mRNA水平)；Ⅱ用SeV刺激Hela细胞0,8,12,24,36h,Western blot检测USP4的表达(蛋白水平)。
1.2在小鼠原代腹腔巨噬细胞中检测RIG-Ⅰ活化对USP4表达的影响。提取小鼠原代腹腔巨噬细胞,
ⅠSeV刺激0,4,8,12,24h,RT-PCR检测USP4的表达(mRNA水平)；
ⅡSeV刺激0,12,24,36h,Western blot检测USP4的表达(蛋白水平)。
2、表达变化的USP4对RIG-Ⅰ介导的IFN-β表达的影响。
2.1mRNA水平检测表达变化的USP4对RIG-Ⅰ介导的IFN-β表达的影响。
a.在HEK293细胞中分别转染空白质粒或USP4的高表达质粒24h后,再分别转染poly(I:C)或poly(dA:dT)12h,RT-PCR检测IFN-β的表达。
b.Ⅰ在HEK293或Hela细胞中分别转染Ctrl siRN八或USI'4siRNA36h后,再转染poly(I:C)0,4h,811,RT-PCR检测IFN-β的表达。Ⅱ在HEK293或Hela细胞中分别转染Ctrl siRN八或USP4siRNA36h后,再转染poly(dA:dT)0,16h,RT-PCR检测IFN-β的表达。Ⅲ在Hela细胞中分别转染Ctrl siRN八或USP4siRNA36h后,SeV刺激0,12h,RT-PCR检测IFN-β的表达。
2.2检测表达变化的USP4对RIG-Ⅰ介导的IFN-β和IRF3报告基因活性的影响。
a.在Hela细胞中分别共转染空白质粒或USP4的高表达质粒和IFN-β、IRF3的报告基因质粒,SeV刺激12h后检测报告基因活性差异。
b.在Hela细胞中分别转染Ctrl siRNA或USP4siRNA,再分别转染IFN-β、 IRF3的报告基因质粒,SeV刺激12h后检测报告基因活性差异。
3、确定USP4的作用靶点。
3.1mRNA水平检测表达变化的USP4对RIG-Ⅰ信号通路中各接头分子介导的IFN-β表达的影响。
a.在HEK293细胞中分别共转染空白质粒或USP4的高表达质粒和RIG-Ⅰ， MAVS, TBK1, IRF35D, TRIF, IKK-ε, MDA5的表达质粒24h, RT-PCR检测IFN-β的表达。
b.在HEK293细胞中分别转染Ctrl siRNA或USP4siRNA24h后,再分别转染RIG-Ⅰ, MAVS, TBK1, IRF35D, MDA5的表达质粒24h, RT-PCR检测IFN-β的表达。
c.在Huh7.5细胞中分别共转染空白质粒或USP4的高表达质粒和RIG-I,MAVS,TBK1,IRF35D的表达质粒24h,RT-PCR检测IFN-β的表达。
3.2检测USP4高表达对RIG-I信号通路中各接头分子介导的IFN-β和IRF3报告基因活性的影响。
在HEK293细胞中分别共转染空白质粒或USP4的高表达质粒和TRIF, RIG-Ⅰ, MAVS, TBK1, IRF35D的表达质粒与IFN-β、IRF3的报告基因质粒,24h后检测报告基因活性差异。
3.3蛋白水平检测表达变化的USP4对各接头分子表达的影响。
a.在HEK293细胞中分别共转染不同剂量的空白质粒或USP4的高表达质粒和RIG-Ⅰ, MAVS, STING TBK1, IRF3, IKK-ε, MDA5的表达质粒,Western Blot检测各接头分子的表达情况。
b.在Hela细胞中分别转染Ctrl siRNA或USP4siRNA36h, Western Blot检测内源性的RIG-Ⅰ, MAVS和TBK1的表达情况。
4、USP4调控RIG-I信号通路的分子机制研究。
4.1根据前面的结果,免疫共沉淀明确USP4与RIG-Ⅰ、MAVS、TBK1等相关靶分子的结合情况。
a.在HEK293细胞中分别共转染带有Myc标签的USP4质粒和带有Flag标签的RIG-Ⅰ、MAVS等质粒,分别用相应抗体进行免疫沉淀检测其相互结合情况。
b.在Hela细胞中转染poly(I:C)0,8,12h,免疫沉淀结合Western Blot检测内源性的USP4与RIG-Ⅰ、MAVS等信号分子的结合情况。
c.在HEK293细胞中分别共转染USP4各节段质粒和RIG-Ⅰ、MAVS等质粒,免疫共沉淀检测各节段与靶分子的结合情况,明确USP4与靶分子结合的结构域。
4.2USP4对相应靶分子泛素化的影响。
a.靶分子表达质粒、USP4表达质粒或空白质粒分别与泛素质粒共转染HEK293细胞,免疫共沉淀结合Western Blot检测USP4对靶分子泛素化的影响。
b.靶分子表达质粒、USP4表达质粒或空白质粒分别与泛素质粒野生型或突变体(K48突变体、K63突变体)共转染HEK293细胞,免疫共沉淀结合Western Blot检测USP4对靶分子泛素化形成(K48或K63)的影响。
c.在HEK293细胞中分别共转染Ctrl siRNA或USP4siRNA后,再共转染靶分子表达质粒和泛素质粒野生型或突变体(K48突变体、K63突变体),免疫共沉淀结合Western Blot检测USP4对靶分子泛素化形式(K48或K63)的影响。
d.在Hela细胞中分别转染Ctrl siRNA或USP4siRNA, SeV刺激4h后,免疫共沉淀结合Western Blot检测USP4对内源性靶分子泛素化的影响,同时明确泛素化形式(K48或K63)。
5、USP4在抗病毒感染中的功能研究。
a.在HEK293细胞中分别转染空白质粒或USP4的高表达质粒24h后,用VSV感染细胞,检测VSV的复制情况。(空斑形成实验检测病毒滴度；实时定量PCR检测细胞中VSV mRNA)
b.在HEK293细胞中分别共转染Ctrl siRNA或USP4siRNA48h后,用VSV感染细胞,检测VSV的复制情况。(空斑形成实验检测病毒滴度,实时定量PCR检测细胞中VSV mRNA)
研究结果：
1、RIG-Ⅰ信号通路的活化能够抑制USP4的表达。
a.用poly(I:C)转染或SeV刺激Hela细胞以激活RIG-Ⅰ信号通路后,USP4的表达被明显抑制(mRNA水平和蛋白水平)。
b.用SeV刺激小鼠原代腹腔巨噬细胞,USP4的表达也明显降低(mRNA水平和蛋白水平)。
2、USP4可以显著增强RIG-Ⅰ介导的IFN-β的表达。
a.在HEK293细胞中转染USP4的高表达质粒后,转染poly(I:C)或poly(dA：dT)(活化RIG-Ⅰ信号通路)诱导的IFN-β表达明显上调。
b.特异性小干扰RNA抑制HEK293细胞或Hela细胞中内源性USP4的表达,转染poly(I:C)或poly(dA:dT)诱导的IFN-β表达被明显抑制。
c.特异性小干扰RNA抑制Hela细胞中内源性USP4的表达,可显著抑制SeV诱导的IFN-β的表达。
d.在Hela细胞中,USP4的高表达可明显增强SeV介导的IFN-β和IRF3报告基因的活化；特异性小干扰RNA抑制USP4的表达后,SeV介导的IFN-β和IRF3报告基因活化被显著抑制。
3、USP4靶向作用于RIG-Ⅰ。
3.1USP4特异性调控RIG-Ⅰ介导的IFN-β表达(mRNA水平)。
a.USP4的过表达可明显增强HEK293细胞中RIG-Ⅰ介导的IFN-β表达(mRNA水平),而对其下游信号分子MAVS, STING, TBK1和接头蛋白MDA5介导的IFN-β的表达无明显影响。在RIG-Ⅰ缺陷的Huh7.5细胞中结果类似。
b.特异性小干扰RNA抑制HEK293细胞中内源性USP4的表达,可显著抑制RIG-Ⅰ介导的IFN-β表达(mRNA水平),但对其下游信号分子MAVS, STING, TBK1, IRF3, IKK-ε和接头蛋白MDA5介导的IFN-β的表达作用不大。
3.2USP4特异性调控RIG-Ⅰ介导IFN-β及IRF3的报告基因活化。
USP4的过表达可明显增强HEK293细胞中RIG-Ⅰ介导的IFN-β及IRF3的报告基因活化,而对其下游信号分子MAVS，TBK1，IRF3及接头分子TRIF介导的IFN-β及IRF3的报告基因活化无明显作用。
3.3USP4特异性增强RIG-Ⅰ的表达。
a.在HEK293细胞中,USP4的过表达能够显著上调外源性RIG-Ⅰ的表达,且具有剂量依赖性；而对于外源性MAVS,STING、TBK1,IRF3,IKK-ε, MDA5的表达无明显影响。
b.特异性小干扰RNA抑制Hela细胞中内源性USP4的表达,可显著并抑制内源性RIG-Ⅰ的表达,而对内源州MAVS.TBK1的表达无明显作用。
4.USP4可以特异性与RIG-Ⅰ相结合。
4.1USP4可以与RIG-Ⅰ相互结合。
a.在HEK293细胞中共转染带有不同标签的USP4和RIG-Ⅰ的表达质粒,分别用相应抗体进行免疫共沉淀,Western Blot的结果显示,外源性的USP4和RIG-Ⅰ可以相互结合。
b.在Hela细胞中，转染poly(I:C)不同的时间点后,用内源性的抗体进行免疫共沉淀,Western Blot的结果显示,内源性的USP4和RIG-Ⅰ能够组成性结合。
4.2USP4特异性结合于RIG-Ⅰ的N末端Card结构域。
在HEK293细胞中分别共转染空白质粒或USP4表达质粒与RIG-Ⅰ仅有N末端两个Card结构域的质粒RIG-IN,Western Blot的结果显示,USP4的过表达可以显著增强RIG-IN的表达；免疫共沉淀结果表明,USP4可以直接与RIG-IN相结合。
4.3USP4与RIG-Ⅰ的结合位点位于其N末端的DUSP结构域。
在HEK293细胞中分别共转染USP4的不同节段质粒(WT，野生型；N260,C末端USP结构域缺失：C261,N末端DUSP结构域缺失)与RIG-Ⅰ表达质粒,免疫共沉淀结果表明,RIG-Ⅰ与WT,N260可以相结合,但是与C261末检测到结合。
5.USP4可以特异性移除RIG-Ⅰ的K48-偶联的泛素链。
5.1USP4的过表达可特异性下调RIG-ⅠK48-偶联的体外泛素化作用。
a. RIG-Ⅰ表达质粒、USP4表达质粒或空白质粒分别与泛素粒共转染HEK293细胞,免疫共沉淀结果表明,USP4过表达能明显下调RIG-Ⅰ的体外泛素化作用。
b. RIG-Ⅰ表达质粒、USP4表达质粒或空白质粒分别与泛素质粒野生型或突变体(K48突变体、K63突变体)共转染HEK293细胞,免疫共沉淀结果显示,USP4过表达可特异性下调RIG-Ⅰ的K48偶联的泛素化。同时,USP4对于MAVS的体外泛素化作用无明显影响。
5.2特异性小干扰RNA抑制HEK293细胞中内源性USP4的表达,可显著上调RIG-ⅠK48-偶联的体外泛素化作用。
a.在HEK293细胞中分别共转染Ctrl siRNA或USP4siRNA后,再共转染RIG-I表达质粒和泛素质粒野生型或突变体(K48突变体、K63突变体),免疫共沉淀结果显示,USP4的低表达能特异性增强RIG-Ⅰ K48-偶联的体外泛素化作用。
b.小干扰RNA抑制Hela细胞中内源性USP4的表达,可显著上调SeV刺激诱导的RIG-Ⅰ内源性泛素化作用,同时其K48-偶联的泛素化作用也明显增强。
6.USP4参与细胞抗病毒反应。USP4的过表达能明显抑制VSV病毒在HEK293细胞中的复制；而用小干扰RNA抑制内源性USP4的表达后能显著促进VSV病毒在胞内的复制。
结论：
1、RIG-Ⅰ信号通路的活化能够抑制USP4的表达。
2、USP4能特异性增强RIG-Ⅰ介导的IFN-β的表达。
3、USP4可以与RIG-β相结合并通过去泛素化酶活性去除其K48-偶联的泛素链来稳定RIG-β的表达,从而正向调节其下游信号通路。
4、USP4参与细胞抗病毒反应。
创新点及意义：
1、本研究首次提出,USP4作为RIG-Ⅰ的一个新的调节分子,通过其去泛素化酶活性去除RIG-Ⅰ K48偶联的泛素链来稳定RIG-Ⅰ的表达,从而正向调节其下游信号通路。
2、本研究为正向调控RLR信号通路提供新的实验证据，为抗病毒研究、诊断和治疗提供新的思路。
Type I IFNs (IFN-a/(3) possess strong anti-viral activity and play pivotal roles in defense against viral infection. During viral infection, pattern-recognition receptors (PRRs), including TLRs and retinoic acid-inducible gene-Ⅰ (RIG-I)-like helicases (RLRs), activate immune cells to produce type IIFN and proinflammatory cytokines, which are involved in the elimination of virus.
Protein ubiquitination plays an essential role in the regulation of RIG-I activation and antiviral immune response. However, the function of the opposite process deubiquitination in RIG-Ⅰ activation remains elusive. In this study, we have identified the deubiquitinating enzyme Ubiquitin-specific protease4(USP4) as a new regulator for RIG-I activation through deubiquitination and stabilization of RIG-Ⅰ. USP4expression was attenuated after virus-induced RIG-Ⅰ activation. Overexpression of USP4significantly enhanced RIG-Ⅰ protein expression and RIG-Ⅰ-triggered IFN-β signaling, at the same time, inhibited VSV replication. siRNA knockdown of USP4expression has an opposite effect. Furthermore, USP4was found to interact with RIG-Ⅰ and remove K48-linked polyubiquitinattion chains from RIG-Ⅰ.
Therefore, our study identified USP4as a new positive regulator for RIG-Ⅰ through deubiquitinating K48-linked ubiquitin chains and stabilizing RIG-Ⅰ.
Objectives:
1. To investigate the function of USP4in RIG-I signaling;
2. To determine the molecular mechanisms and targets of USP4;
3. To investigate the effect of USP4on antiviral response.
Methods:
1. Identification differentially expressed USP4upon RIG-I activation in different cell lines.
1.1Identification differentially expressed USP4upon RIG-I activation in Hela cells.
a. I RT-PCR analysis of USP4expression in Mela cells transfected with poly(I:C) for0,2,4,8,12h (mRNA level);
II Western blot analysis of USP4expression in Mela cells transfected with poly(I:C) for0,12,24,36h (protein level).
b. I RT-PCR analysis of USP4expression in Hela cells infected with SeV (Sendai virus) for0,8,12,24h (mRNA level);
II Western blot analysis of USP4expression in Mela cells infected with SeV for0,8,12,24,36h (protein level).
1.2Identification differentially expressed USP4upon RIG-I activation in mouse peritoneal macrophages.
After bone marrow derived macrophages (BMDM) were infected with SeV for0,4,8,12,24h, RT-PCR analysis of USP4expression (mRNA level); infected with SeV for0,12,24,36h. Western blot analysis of USP4expression (protein level).
2. Confirmation of whether the RIG-I-induced IFN-β production was regulated by USP4.
2.1RT-PCR analysis whether the RIG-I-induced IFN-β production was regulated by USP4on mRNA level.
a. HEK293cells transfected with the indicated plasmids were transfected with poly(I:C) or poly(dA:dT) for12h. Total RNA was prepared and analyzed for the expressions of IFN-β, USP4and GAPDH by RT-PCR.
b.ⅠHEK293or Hela cells were transfected with control siRNA or USP4siRNA for36h, and then transfected with poly(I:C) or for0,4h,8h, Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.
Ⅱ HEK293or Hela cells were transfected with control siRNA or USP4siRNA for36h, and then transfected with poly(dA:dT) or for0,16h, Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.
Ⅲ Hela cells were transfected with control siRNA or USP4siRNA for36h, and then infected with SeV for12h. Total RNA was prepared and analyzed for the expressions of IFN-β and GAPDH by RT-PCR.
2.2Luciferase assay analysis whether the RIG-I-induced IFN-P promoter and IRF3activation was regulated by USP4.
a. Hela cells were transiently transfected with IFN-β or IRF3reporter plasmid together with USP4expression plasmid or control plasmid, analyzed luciferase activity after SeV infection for12h.
b. Hela cells were transfected with control siRNA or USP4siRNA, and then transfected with IFN-P or IRF3reporter plasmid, analyzed luciferase activity after SeV infection for12h.
3、To determine the molecular targets of USP4.
3.1RT-PCR analysis the IFN-β production induced by RIG-I or adaptors in RIG-I pathway on mRNA level modulated upon differentially expression of USP4.
a. HEK293cells were transfected with RIG-I, MAVS, TBK1, IRF35D, TRIF, IKK-e, MDA5along with USP4plasmid, and24h later IFN-P mRNA were analyzed by RT-PCR.
b. HEK293cells were transfected with RIG-I, MAVS, TBK1, IRF35D, MDA5along with USP4specific siRNA or control siRNA, and IFN-P mRNA were analyzed by RT-PCR. c. Huh7.5cells were transfected with RIG-I, MAVS, IRF35D along with USP4plasmid, and24h later IFN-β mRNA were analyzed by RT-PCR.
3.2Luciferase assay analysis the RIG-I or other adaptors in RIG-I pathway induced IFN-β promoter and IRF3activation was regulated upon overexpression of USP4.
HEK293cells were transfected with TRIF, RIG-I, MAVS, TBK1, IRF35D along with IFN-β or IRF3reporter plasmid and USP4plasmid, and24h later luciferase activity was analyzed.
3.3Western blot analysis of expression of adaptors in RIG-I pathway upon differentially expression of USP4.
a. Western blot analysis of the lysates from HEK293cells transfected with Myc-RIG-I, MAVS, STING, TBK1, IRF3, IKK-ε, MDA5together with increasing concentration of Flag-USP4expression plasmid.
b. Western blot analysis of the expression of RIG-I, MAVS, and TBK1in Hela cells transfected with control siRNA or USP4siRNA and36h later infected with SeV for indicated time periods.
4. To clarify the molecular mechanisms of USP4.
4.1To investigate the interaction between USP4and the target molecules by immunopreicpitation.
a. Lysates from HEK293cells transiently cotransfected with Flag-tag target molecules and Myc-USP4expression plasmids were subjected to immunoprecipitation with anti-Myc or anti-Flag antibody followed by western blot analysis with anti-Flag or anti-Myc antibody.
b. Hela cells transfected with poly(I:C) for0,8,12h were subjected to immunoprecipitation with anti-USP4or control IgG followed by western blot analysis with specific antibody. Proteins in whole-cell lysate were used as positive control (Input).
c. Lysates from HEK293cells transiently cotransfected with Flag-tagged target molecule and HA-tagged USP4mutants were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.
4.2To test the role of USP4in ubiquitination of the target molecular.
a. Lysates from HEK293cells transiently cotransfected with Flag-target molecule, Myc-USP4and HA-Ub plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.
b. Lysates from HEK293cells transiently cotransfected with Flag-target molecule, Myc-USP4or vector control and HA-Ub (WT), HA-Ub (K48) or HA-Ub (K63) plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.
c. Lysates from HEK293cells transfected with control siRNA or USP4siRNA and Flag-target molecule and HA-Ub (WT), HA-Ub (K48) or HA-Ub (K63) plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-HA antibody.
d. Lysates from Hela cells transfected with control siRNA or USP4siRNA and infected with SeV for4h were subjected to immunoprecipitation with specific antibody followed by western blot analysis with anti-Ub or anti-Ub(K48) antibody.5. To investigate the effect of USP4on antiviral response.
a. HEK293cells (2×105) transfected with the indicated plasmids (1μ g each).24h later, cells were further transfected with poly(I:C)(0.1μ g) or left untreated.18h after poly(I:C) transfection, cells were infected with VSV (MO1,0.1), and the supernatants were harvested at12h post-infection. Supernatants were analyzed for VSV titers with standard plaque assays. Intracellular VSV RNA replicates were measured by QRT-PCR.
b. HEK293cells (2×105) were transfected with control siRNA or USP4siRNA and then treated as in a.
Results:
1. USP4expression is inhibited by RIG-Ⅰ activation.
a. Transfection of poly(I:C) or infection of SeV, which has been shown to activate RIG-I signaling substantially attenuated USP4mRNA and protein expression in Hela cells.
b. SeV infection could also attenuate USP4expression in murine peritoneal macrophages.
2. USP4enhances RIG-Ⅰ-induced IFN-β production.
a. In HEK293cells, overexpression of USP4further increased poly(I:C)-induced expression of IFN-β.
b. USP4knockdown through siRNA transfection significantly inhibited poly(I:C) or poly(dA:dT)-induced IFN-β expression in HEK293cells. Similarly, poly(I:C) or poly(dA:dT)-induced IFN-β production was also greatly attenuated by USP4knockdown in Hela cells.
c. SeV-induced IFN-β production was substantially attenuated after knockdown of USP4expression in Hela cells.
d. USP4overexpression significantly enhanced SeV-induced IFN-β promoter and IRF3activation in Hela cells. In contrast, USP4knockdown significantly inhibited SeV-induced IFN-β promoter and IRF3activation.
3. USP4targets RIG-I.
3.1USP4specifically enhanced RIG-Ⅰ-induced IFN-β mRNA expression.
a. In HEK293cells, USP4enhanced RIG-Ⅰ-induced IFN-β mRNA expression, while, MDA5-, MAVS-, TBK1-, IKK-ε-, TRIF-and IRF35D-induced IFN-β mRNA expression was not impaired by USP4overexpression. Similarly, RIG-Ⅰ-induced IFN-β mRNA expression was also attenuated by USP4overexpression in Huh7.5cells.
b. In HEK293cells, USP4siRNA transfection substantially decreased RIG-induced IFN-β mRNA expression, but not MDA5-, MAVS-, TBK1-, IKK-ε-, TRIF and IRF35D-induced IFN-β mRNA expression.
3.2USP4specifically enhanced RIG-Ⅰ-induced activation of IFN-β promoter and IRF3reporter.
RIG-Ⅰ-induced activation of IFN-β promoter and IRF3reporter were also significantly enhanced by USP4overexpression. But, MAVS-, TBK1, TRIF and IRF3-induced activation of IFN-β promoter and IRF3reporter was not affected by USP4ovexpression.
3.3USP4specifically enhanced RIG-Ⅰ expression.
a. USP4expression significantly enhanced RIG-Ⅰ expression in a dose dependent manner. In contrast, USP4overexpression had no effects on MDA5, MAVS, STING, TBK1, IKK-ε and IRF3expression.
b. Knockdown of USP4expression significantly decreased RIG-Ⅰ protein expression especially. As a control, MAVS and TBK1protein levels were not impaired.
4. USP4specifically interacts with RIG-Ⅰ.
4.1USP4interacts with RIG-Ⅰ.
a. Myc-USP4and Flag-RIG-I were cotransfected into HEK293cells,24h after transfection, immunoprecipitation experiments were performed with Myc or Flag antibodies. Flag-RIG-I was coprecipitated with Myc-USP4using anti-Myc antibody and vice versa.
b. Endogenous interaction between RIG-Ⅰ and USP4was examined in Hela cells transfected with poly(I:C) for various times by immunopreicpitation with USP4antibody and western blotting with RIG-Ⅰ antibody. USP4was found constitutively to interact with RIG-Ⅰ. As a control, the interaction could not be detected with normal IgG.
4.2USP4interacts with the N-terminal domain of RIG-Ⅰ.
Two RIG-Ⅰ truncations (N-terminal domain composed of two CARD motifs and a C-terminal domain) were used in the immunoprecipitation assays. USP4was found to interact with the N-terminal domain of RIG-Ⅰ. Consistently, overexpression of USP4could also increase the protein expression of the RIG-Ⅰ mutant RIG-Ⅰ-N harboring the N-terminal domain.
4.3USP4and RIG-Ⅰ form a complex through the DUSP domain of USP4and the N-terminal domain of RIG-Ⅰ.
Coimmunoprecipitation experiments showed that RIG-Ⅰ interacted with WT USP4and the N-terminal DUSP domain, but not the C-terminal USP domain.
5. USP4removes K48-linked polyubiquitination conjugate from RIG-I.
5.1USP4overexpression remarkably decreased RIG-Ⅰ K48-linked ubiquitination.
a. RIG-Ⅰ was co-expressed with HA-ubiquitin and USP4or vector control. Cotransfection of USP4expression plasmid remarkably decreased RIG-Ⅰ ubiquitination.
b. Ubiquitin mutant vectors K48and K63, which contain arginine substitutions of all of its lysine residues except the one at position48and63respectively, were used in the transfection assays. Cotransfection of USP4greatly decreased the polyubiqutination of RIG-Ⅰ in the setting of ubiquitin WT and K48transfected cells, but not in the K63-transfected cells. In contrast, neither K48-linked nor K63-linked ubiquitination of MAVS was impaired by USP4overexpression.
5.2K-48-linked polyubiqutination of RIG-I was greatly increased after USP4knockdown.
a. The level of RIG-Ⅰ polyubiqutination was greatly increased after transfection of USP4siRNA in the setting of WT ubiquitin and ubiquitin mutant K.48transfected cells compared to control siRNA transfected cells, but not in mutant K63transfected cells.
b. Transfection of USP4siRNA further increased K48-linked polyubiqutination of RIG-Ⅰ in SeV-transfected Mela cells.
6. USP4is involved in cellular antiviral response.
Plaque assays of HEK.293cells infected with VSV showed that overexpression of USP4substantially decreased viral replication compared to control vector-transfected cells. And transfection of USP4siRNA greatly increased VSV viral replication in HEK.293cells.
Conclusion:
1、USP4expression is inhibited by RIG-I activation.
2、USP4enhances RIG-I-induced IFN-β production.
3、USP4positively regulates RIG-I pathway by interacting with RIG-I and removing K48-linked polyubiquitinattion chains from RIG-I.
4、USP4is involved in cellular antiviral response.
Innovation and significances:
1. We identified USP4as a new positive regulator for RIG-I through deubiquitinating K48-linked ubiquitin chains and stabilizing RIG-I.
2. Our study provided new experimental evidence for negative regulation anti-virus research.
引文
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