Fas-Associated Factor 1 Negatively Regulates Antiviral Immune Response by Inhibiting ...

48 Pages·2016·2.23 MB·English

MCB Accepted Manuscript Posted Online 25 January 2016
Mol. Cell. Biol. doi:10.1128/MCB.00744-15
Copyright © 2016 Song et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Fas-Associated Factor 1 Negatively Regulates Antiviral
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Immune Response by Inhibiting Translocation of IRF3 to
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the Nucleus
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5 Soonhwa Song, Jae-Jin Lee, Hee-Jung Kim, Jeong Yoon Lee, Jun Chang and
6 Kong-Joo Lee
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8 From the Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans n
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9 University, Seoul, Korea 120-750 a
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11 * Running title: FAF1 inhibits virus induced innate immunity. fr
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13 To whom correspondence should be addressed: Kong-Joo Lee, Ph. D, Graduate School of tt
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14 Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea, //
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15 Tel.: [phone]; Fax: [phone]; E-mail: [email protected] c
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21 Abstract
22 This study is designed to examine the cellular functions of human Fas-associated factor 1
23 (FAF1) containing multiple ubiquitin-related domains. Microarray analyses revealed that
24 interferon stimulated genes related to antiviral response are significantly increased in FAF1
25 knocked down HeLa cells. Silencing FAF1 enhanced the poly I:C and respiratory syncytial D
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26 virus (RSV) induced productions of type I interferons (IFNs), the target gene of interferon n
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27 regulator factor 3 (IRF3). IRF3 is a key transcription factor in IFNβ signaling responsible for d
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28 host innate immune response. This study also found that FAF1 and IRF3 physically associate f
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29 with IPO5/importin-β3 and that overexpression of FAF1 reduces the interaction between
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30 IRF3 and IPO5/importin-β3. These findings suggest that FAF1 negatively regulates IRF3- p
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31 mediated IFNβ production and antiviral innate immune response by regulating nuclear c
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32 translocation of IRF3. We conclude that FAF1 plays a novel role in negatively regulating a
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33 virus-induced IFNβ production and the antiviral response by inhibiting the translocation of .o
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34 active phosphorylated IRF3 from cytosol to nucleus. /
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37 Introduction
38 Innate immune system, in contrast to adoptive immune response present only in immune cells,
39 is present in all cells and plays key roles in host defense against viral infections by sensing
40 and immediately responding to the invading pathogens (1, 2). Intracellular pattern recognition
41 receptors (PRRs) including Toll like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)- D
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42 like receptors (RLRs) and nucleotide-binding oligomerization domain containing (NOD)-like n
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43 receptors (NLRs) recognize pathogen associated molecular patterns (PAMPs) and activate d
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44 innate immune signaling pathways leading to the production of type I interferons (IFNα/β) f
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45 and other cytokines. Type I IFNs play crucial role in limiting viral replication and priming
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46 adaptive immune response (3, 4). IFNβ can be produced in most cell types, and when the p
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47 cells are infected with a virus, IFNβ expression rapidly increases due to the activation of c
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48 transcription factors (5). Transcription factor complexes including interferon regulatory factor a
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49 3 (IRF3), nuclear factor kappa B (NF-κB) and AP1 are bound to the regulatory domains of .o
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50 IFNβ promoter and cooperatively regulate the transcription of IFNβ (6). IFNβ secreted from /
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51 infected cells, binds to IFNAR1/2 on adjacent cells and then activates JAK/STAT signaling M
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52 pathway, which results in the expression of interferon stimulated genes (ISGs). Some ISGs c
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53 such as Mx1, OAS1 and IFIT1 directly interfere viral replication, while others including ,
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54 RIG-I, MDA5 and IRF7 indirectly do so by enhancing IFNβ production (7). 1
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55 The transcription factor, IRF3, plays the most critical role in the regulation of virus- g
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56 induced IFNβ activation. IRF3 is constitutively expressed and localized in cytoplasm in a s
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57 latent form. Single stranded or double stranded viral RNAs accumulated inside cells after
58 infection are recognized by RLRs and TLR3 which recruit adaptor proteins MAVS and TRIF
59 respectively (8, 9). These adaptor proteins, MAVS and TRIF, recruit the kinases TBK1 and
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60 IKKε, which activate IRF3 by phosphorylating C-terminal region of IRF3 at seven Ser/Thr
61 residues (Ser385, 386, 396, 398, 402, 405 and Thr404). Phosphorylated IRF3 forms dimers
62 which shuttle into the nucleus, where they interact with co-activator CBP/p300 and initiate
63 transcription of target genes including IFNβ (10, 11). It has been reported that
64 phosphorylation of IRF3 at Ser386 induces dimerization and interaction with CBP (11), and D
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65 that phosphorylation at Ser396 occurs in response to viral infections (10). Mutation studies w
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66 confirmed that phosphorylations at Ser386 and Ser396 are important for IRF3 activation and a
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67 interaction with CBP (12). d

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68 The production of IFNβ is essential for protecting cells from virus infection, and
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69 aberrant activation of IFNβ production can trigger diseases such as multiple sclerosis and p
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70 systemic lupus erythematosus (SLE) (13, 14). Therefore, IFNβ production needs to be tightly c
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71 regulated. Several positive and negative regulators have been identified. Studies of a
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72 mechanisms in IRF3 activation as well as in the negative regulation of transcriptional activity .o
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73 of IRF3 are still on-going. The two negative regulatory mechanisms so far identified as /
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74 already noted, are degradation of IRF3 following its phosphorylation by ubiquitin proteasome M
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75 system and posttranslational modifications of IRF3 which inhibit its activity. RAUL, a major c
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76 ubiquitin E3 ligase, ubiquitinates IRF3 regardless its phosphorylation status (15), while E3 ,
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77 ubiquitin ligase RBCK1 and cytoplasmic peptidyl-prolyl-isomerase Pin1 ubiquitinate only 1
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78 phosphorylated IRF3, and trigger its degradation (16, 17). The second negative regulation y

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79 mechanism reported to change IRF3 activity is post-translational modification of IRF3. e
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80 Protein phosphatase 2A (PP2A) and MAPK phosphatase 5 (MKP5) are known to
81 dephosphorylate IRF3 and decrease IFN response (18, 19). SUMOylations of IRF3 are
82 another known mechanism to decrease IRF3 activity (20).
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83 Thus phosphorylation is an indispensable step for IRF3 activation, and phosphorylated
84 IRF3 is translocated into the nucleus to bind IFNβ promoter. However, the mechanism
85 underlying the translocation process remains elusive. Previous studies demonstrated that
86 IRF3 has an active nuclear localization signal (NLS) which is recognized by importin-α
87 receptors and transported to the nucleus (21, 22). IRF3 also has an active nuclear export D
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88 signal (NES), it is exported from nucleus via CRM1-mediated pathway to mainly localize in w
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89 the cytoplasm in un-stimulated cells (23). Following infection, IRF3 resides in the nucleus a
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90 and interacts with CBP (21, 24). This study reports that FAF1 as a negative regulator of virus d

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91 triggered IFNβ signaling pathway by inhibiting the nuclear translocation of phosphorylated m

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92 IRF3. tt
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93 Fas-associated factor 1 (FAF1) was first identified as a component of apoptosis signaling c
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94 pathway (25). FAF1 is an ubiquitin receptor containing multiple ubiquitin related domains a
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95 that include an ubiquitin-associated (UBA) domain and three domains with ubiquitin-like .o
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96 folds, UBL1, UBL2, and ubiquitin regulatory X (UBX) (26). N-terminal UBA domain (47 /
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97 amino acids long) recruits Lys48-linkage polyubiquitinated proteins required for FAF1- M
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98 mediated apoptosis and stress response (26, 27). UBL1 domain binds to Hsp70 and regulates c
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99 its chaperone activity by promoting Hsp70 degradation (28, 29). C-terminal UBX domain ,
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100 interacts only with valosin-containing protein (VCP, AAA ATPase p97) complexed with 1
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101 Npl4-Ufd1 heterodimer. This interaction regulates the binding of the polyubiquitinated y

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102 proteins via N-terminal UBA domain. FAF1 promotes the degradation of ERAD substrate in e
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103 a VCP-Npl4-Ufd1-dependent manner (26, 30). To further understand the cellular functions of
104 FAF1, we investigated the target genes of FAF1 using microarray analysis. This investigation
105 reveals that FAF1 is involved in negative regulation of virus triggered IFNβ signaling
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106 pathway, in a novel manner by inhibiting the nuclear translocation of phosphorylated IRF3,
107 subsequently IFNβ production, and thereby inhibiting cellular antiviral response.
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108 Materials and Methods
109 Reagents and plasmids
110 The following are the sources of antibodies used in this study: mouse monoclonal Flag
111 antibody was from Sigma (MO, USA); rabbit anti-IRF3, mouse anti-MAVS, mouse anti-
112 tubulin, rabbit anti-HDAC1 and mouse anti-actin antibodies were from Santa Cruz D
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113 Biotechnology (CA, USA); rabbit anti-Mx1 and rabbit anti-phospho-IRF3 (Ser386) were n
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114 from Abcam (Cambridge, UK); rabbit anti-FAF1 and rabbit anti-GAPDH were from ad
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115 AbFrontier (Seoul, Korea); rabbit antibodies specific for IRF3, phospho-IRF3 (Ser396),
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116 TBK1, TRIF, ISG15 and STAT1 were from Cell signaling Technology (MA, USA); mouse m

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117 anti-GFP antibody was from Life technologies (CA, USA); mouse anti-RIG-I antibody was tp
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118 from Adipogen AG (CA, USA); mouse antibodies specific for IPO5 and FAF1 from Abnova m
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119 (CA, USA). Cycloheximide (C7698) and leptomycin B (L2913) were purchased from Sigma .a
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120 (MO, USA). .
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121 pISRE-Luc was provided by Dr. Greg Barton (University of California-Berkeley, USA). o
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122 pIFNB-GL3 and pIFNA4-GL3 were provided by Dr. John Hiscott (Vaccine & Gene Therapy M
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123 Institute of Florida, USA) and pCMV-beta-gal was from Dr. Eunsuk Hwang (Ewha Womans h

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124 University, Korea). The expression plasmids for TRIF, IRF3-WT-GFP and IRF3-5D-GFP , 2
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125 were kindly provided by Dr. Joo Young Lee (Catholic University of Korea) with kind 9

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126 permission from Dr. Katherine A. Fitzgerald (University of Massachusetts Medical School, y
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127 USA) and Dr. John Hiscott (Vaccine & Gene Therapy Institute of Florida, USA). Flag-RIG-I e
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128 N was from Dr. Takashi Fujita (Tokyo, Japan). Flag-MAVS and Flag-TBK1 were from Dr.
129 Glen N. Barber (University of Miami Miller School of Medicine, USA). Flag-IKKε was from
130 Dr. Ki-Sun Kwon (KRIBB, Korea). pFlag-CMV-2-FAF1 WT, pFlag-CMV-2-FAF1[82-650],
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131 pFlag-CMV-2-FAF1ΔUBX (a.a.; 1-568), pFlag-CMV-2-FAF1ΔUBL1-2 (a.a.; Δ100-270),
132 pFlag-CMV-2-FAF1ΔUAS (a.a.; Δ352-487) and pFlag-CMV-2-FAF1[1-201] were prepared
133 as previously described (26, 27). pFlag-CMV-2-FAF1[1-351] was generated by cloning. All
134 plasmid constructs were verified by DNA sequencing.
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136 Cell culture and transfection lo
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137 HeLa cells were purchased from ATCC and cultured in EMEM supplemented with 10% fetal d

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138 bovine serum, 100 units/mL of penicillin G and 100 μg/mM streptomycin at 37°C in a 5% m

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139 CO2-containing humidified incubator. HEK293T cells and Raw264.7 cells were cultured in tt
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140 DMEM supplemented under same conditions. HEp-2 cells were cultured in MEM /m
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141 supplemented under same conditions. For transient overexpression of specific proteins, cells b.
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142 were transfected using LT-1 and analyzed at 24 h or 48 h post transfection. For gene silencing, m
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143 FAF1 siRNAs were obtained from Dharmacon (ON-TARGETplus SMARTpool siRNA, IL, g
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144 USA) and Bioneer (Daejeon, Korea). FAF1 siRNA #1 was from Dharmacon (L-009106-00- n

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145 0005) and FAF1 siRNA #2 (Cat No. 1049605) and control siRNA were from Bioneer. siRNA ar
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146 #2 targets consensus sequence in both human and mouse FAF1. Cells were transfected with 8
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147 siRNAs using DharmaFECT1 following manufacturer’s protocol at a final concentration of 0
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148 100 nM. b
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149 e
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150 Microarray analysis
151 HeLa cells were transfected with FAF1 or control siRNA and collected at 48 h post
152 transfection. Total RNA was extracted using Trizol (Invitrogen Life Technologies, CA, USA),
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153 purified using RNeasy columns (Qiagen, CA, USA) according to the manufacturers’ protocol.
154 For quality control, RNA purity and integrity were evaluated by denaturing gel
155 electrophoresis, OD 260/280 ratio, and analyzed on Agilent 2100 Bioanalyzer (Agilent
156 Technologies, CA, USA). Total RNA was amplified and purified using the Ambion Illumina
157 RNA amplification kit (Ambion, CA, USA) to yield biotinylated cRNA according to the D
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158 manufacturer’s instructions. Briefly, 550 ng of total RNA was reverse-transcribed to cDNA w
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159 using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed, and a
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160 labeled with biotin-NTP. After purification, the cDNA was quantified using the ND-1000 d

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161 Spectrophotometer (NanoDrop, Wilmington, USA). 750 ng of labeled cDNA samples were m

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162 hybridized to each Human HT12 expression v.4 bead array for 16-18 h at 58°C, according to tt
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163 the manufacturer's instructions (Illumina, Inc., San Diego, USA). Array signals were detected /m
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164 using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, .
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165 UK) following the bead array manual. Arrays were scanned with an Illumina bead array
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166 Reader confocal scanner according to the manufacturer's instructions. The quality of g
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167 hybridization and overall chip performance were monitored by visual inspection of both
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168 internal quality control checks and the raw scanned data. Raw data were extracted using the r
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169 software provided by the manufacturer (Illumina GenomeStudio v2011.1 (Gene Expression 8
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170 Module v1.9.0)). Probes signal value was transformed by logarithm and normalized by 0
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171 quantile method. Statistical significance of the expression data was determined using LPE b
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172 test and Fold change in which the null hypothesis was that no difference exists among 2 u
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173 groups. False discovery rate (FDR) was controlled by adjusting p value using Benjamini- t
174 Hochberg algorithm.
175
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176 Microarray data accession number
177 The raw and processed microarray data are available in the GEO public database under GEO
178 accession number ‘GSE71665’ (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE71665)
179
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180 Luciferase reporter assay w
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181 The cells were transfected with reporter genes and pCMV-beta-gal, then after 24 h, poly I:C d
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182 (10 μg/mL) was treated by transfection. Transfected cells were harvested and luciferase
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183 activity and beta-galactosidase activity were measured using Luciferase assay system m

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184 (Promega, WI, USA) and Galacto-light plus system (Applied Biosystems, CA, USA) tp
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185 respectively on luminometer (Luminoskan TL plus, ThermoFisher Scientific, MA, USA).
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186 Each experiment was repeated in triplicate and firefly luciferase activities were normalized to .a
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187 beta-galactosidase activities. .
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188 o
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189 Quantitative RT-PCR a
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190 Cells were harvested at 48 h post transfection. Total RNA from these cells was isolated using 8
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191 RNeasy mini kit (Qiagen, CA, USA) then reverse transcribed using SuperScript Ⅱ RT 01
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192 (Invitrogen Life Technologies, CA, USA) according to manufacturer’s protocol. Synthesized y

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193 cDNA was subjected to real time PCR (AB7300, Applied Biosystems, USA) for e
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194 amplification in triplicate. PCR reactions were performed using SYBR Green qPCR Master
195 Mix (Applied Biosystems, CA, USA) and specific primers: hIFNβ (31) 5’ CAA CAA GTG
196 TCT CCT CCA AAT -3’ (sense) and 5’-TCT CCT CAG GGA TGT CAA AG-3’ (antisense);
197 mIFNβ 5’-CAT CAA CTA TAA GCA CCA-3’(sense) and 5’-TTC AAG TGG AGA GCA
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