Enterovirus 71 Proteins 2A and 3D Antagonize the Antiviral Activity of IFN-γ via Signaling Attenuation

37 Pages·2015·5.18 MB·English

JVI Accepted Manuscript Posted Online 29 April 2015
J. Virol. doi:10.1128/JVI.00205-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
EV71 Proteins Attenuate IFN-γ Signaling 1
1 Enterovirus 71 Proteins 2A and 3D Antagonize the Antiviral Activity of
2 IFN-γ via Signaling Attenuation
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4 Li-Chiu Wang,a Su-O Chen,b Shih-Ping Chang,c Yi-Ping Lee,a Chun-Keung Yu,a,c
5 Chia-Ling Chen,d Po-Chun Tseng,b Chia-Yuan Hsieh,b Shun-Hua Chen,a,c,e Chiou-Feng D
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6 Lin,f n
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8 Institute of Basic Medical Sciences,a Institute of Clinical Medicine,b and Department of o
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9 Microbiology and Immunology,c College of Medicine, National Cheng Kung University, h
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10 Tainan 701, Taiwan; Center for Translational Medicine, Taipei Medical University, Taipei 110, //
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11 Taiwand; Center of Infectious Disease and Signaling Research, National Cheng Kung i.a
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12 University, Tainan 701, Taiwane; Graduate Institute of Medical Sciences, Department of .
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13 Microbiology and Immunology, College of Medicine, Taipei Medical University, Taipei 110, /

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16 Running Title: EV71 Proteins Attenuate IFN-γ Signaling 1
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18 Keywords: EV71, viral protein, IFN-γ, STAT1, signal transduction s
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20 Address correspondence to Shun-Hua Chen, [email protected] and Chiou-Feng Lin,
21 [email protected]
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EV71 Proteins Attenuate IFN-γ Signaling 2
23 ABSTRACT
24 Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible
25 mechanisms including cytokine storm, brainstem encephalitis, and fulminant pulmonary
26 edema. Interferon (IFN)-γ may confer anti-EV71 activity; however, the claim that disease
27 severity is highly correlated to an increase in IFN-γ is controversial and would indicate an
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28 immune escape initiated by EV71. This study investigating the role of IFN-γ in EV71 o
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29 infection using a murine model showed that IFN-γ was elevated. Moreover, IFN-γ lo
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30 receptor-deficient mice showed higher mortality and more severe disease progression with
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31 slower viral clearance than wild-type mice. In vitro results showed that IFN-γ pre-treatment fr
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32 reduced EV71 yield whereas EV71 infection caused IFN-γ resistance with attenuated IFN-γ
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33 signaling in IFN regulatory factor (IRF) 1 transactivation. To study the immunoediting ability
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34 of EV71 proteins on IFN-γ signaling, 11 viral proteins were stably expressed in cells without i.
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35 cytotoxicity; however, viral proteins 2A and 3D blocked IFN-γ-induced IRF1 transactivation m
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36 following a loss of signal transducer and activator of transcription (STAT) 1 nuclear g
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37 translocation. Viral 3D attenuated IFN-γ signaling accompanied by a STAT1 decrease without n

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38 interfering with IFN-γ receptor expression. Restoration of STAT1 or blocking 3D activity was
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39 able to rescue IFN-γ signaling. Interestingly, viral 2A attenuated IFN-γ signaling using ,

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40 another mechanism by reducing the serine phosphorylation of STAT1 following the 1
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41 inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. y

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42 These results demonstrate the anti-EV71 ability of IFN-γ and the immunoediting ability by
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43 EV71 2A and 3D, which attenuate IFN-γ signaling through different mechanisms.
EV71 Proteins Attenuate IFN-γ Signaling 3
44 Importance
45 Immunosurveillance by interferon (IFN)-γ may confer anti-enterovirus 71 (EV71) activity;
46 however, the claim that disease severity is highly correlated to an increase in IFN-γ is
47 controversial and would indicate an immune escape initiated by EV71. IFN-γ
48 receptor-deficient mice showed higher mortality and more severe disease progression,
49 indicating the anti-EV71 property of IFN-γ. However, EV71 infection caused cellular D
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50 insusceptibility in response to IFN-γ stimulation. We used an in vitro system with viral n
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51 protein expression to explore the novel IFN-γ inhibitory properties of the EV71 2A and 3D
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52 proteins through the different mechanisms. According to this study, targeting either 2A or 3D
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53 pharmacologically and/or genetically may sustain a cellular susceptibility in response to m

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54 IFN-γ, particularly for IFN-γ-mediated anti-EV71 activity. tt
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EV71 Proteins Attenuate IFN-γ Signaling 4
56 Introduction
57 Enterovirus 71 (EV71) is a single-stranded RNA virus in the Picornaviridae family. The
58 EV71 genome encodes four structural proteins, VP1-4, and seven non-structural proteins,
59 2A-C and 3A-D (1). Numerous studies have investigated the functions of viral proteins in
60 viral replication and virulence (2). During EV71 infection, capsid VP proteins mediate virus
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61 entry by binding to cellular receptors, human scavenger receptor class B and P-selectin o
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62 glycoprotein ligand-1 (3). Additionally, VP proteins participate in the assembly of viral lo
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63 particles (4). The 3C protein, a chymotrypsin-like protease, reduces host cell transcription
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64 dramatically by inhibiting cell polyadenylation (5) and induces caspase-regulated neural cell fr
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65 apoptosis (6). To develop specific anti-EV71 drugs, a number of small molecules targeting
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66 viral proteins have been designed, such as the 3C inhibitor, rupintrivir and the 3D inhibitor,
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67 aurintricarboxylic acid (ATA) (7-9). i.
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68 EV71 infection typically causes mild, self-limiting hand-foot-and-mouth disease; however, .
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69 patients sometimes have significant morbidity and mortality resulting from hemorrhagic /

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70 pulmonary edema following acute central nervous system-related cardiopulmonary failure A
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71 and brainstem encephalitis (2, 10, 11). In addition to the direct cytotoxicity caused by EV71 il
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72 infection (12-16) and the resultant virulence factors (6, 17), host factors such as the aberrant 2
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73 production of cytokines that is detected during EV71-associated pulmonary edema can also 9

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74 lead to disease. In infected patients with brainstem encephalitis, increased levels of
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75 interleukin (IL)-8, IL-10, IL-13, interferon (IFN)-γ, CXC chemokine ligand 10
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76 (IFN-γ-inducible protein 10), and monokine induced by IFN-γ are observed in the serum (18,
77 19), and high amounts of IL-1β, IL-6, IL-8, IFN-γ, and chemokine (C-C motif) ligand 2
78 (monocyte chemotactic protein-1) are found in the cerebrospinal fluid (19, 20). Neutralization
79 of IL-6 by antibody confers protection against severe complications in EV71-infected neonate
80 mice (21). Additionally, an increased frequency of Th17 cells in peripheral blood and
EV71 Proteins Attenuate IFN-γ Signaling 5
81 increased serum levels of the Th17 cell-derived cytokines, IL-17 and IL-23 are observed in
82 EV71-infected patients (22). While immunopathogenesis of EV71 infection has been
83 proposed (23), the protective versus the pathogenic effects of most cytokines have not been
84 addressed.
85 Host-derived IFNs exert antiviral activity via several mechanisms, including interference
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86 with viral replication through inhibiting the transcription and translation of viral components o
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87 (24). Studies have demonstrated the antiviral activity of type I IFNs against EV71 infection in lo
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88 vitro and in vivo (25, 26). Furthermore, treatment with an IFN-inducing agent (27) and
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89 synergistic inhibition of EV71 replication by IFN-α and rupintrivir (28) have been identified fr
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90 as effective treatment regimens. However, during EV71 infection, several immune escape
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91 strategies are elicited by EV71 to attenuate the type I IFN response. The viral 3C protein
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92 retards retinoid acid-inducible gene I-mediated IFN regulatory factor (IRF) 3 activation (29) i.
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93 and inhibits Toll-like receptor 3 signaling by cleaving the adaptor proteins TRIF (30) and m
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94 IRF7 (31) to reduce type I IFN production (29-32). Viral protein 2A decreases the type I IFN g
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95 response by targeting the antiviral signaling protein in mitochondria (33) and by reducing the n

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96 IFN-α/β receptor (IFNAR) 1 level (34). In addition to type I IFNs, the role of type II IFN
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97 (IFN-γ), a pro-inflammatory cytokine that is produced by natural killer cells, T cells, and ,

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98 antigen-presenting cells for immune defense against microbial infections and tumorigenesis 1
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99 (35, 36), is controversial in EV71 infection, specifically with regard to its dual roles in both y

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100 cellular protection and immunopathogenesis. Compared to mice with IFNAR deficiency
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101 alone, mice with both IFNAR and IFN-γ receptor (IFN-γR) deficiency display increased
102 susceptibility to EV71 infection (37), indicating a protective role for IFN-γ. In contrast,
103 expression and gene polymorphism of IFN-γ has been shown to associate with EV71-induced
104 pulmonary edema and encephalitis in patients (18, 38), and exogenous administration of three
105 cytokines (IL-6, IL-13, and IFN-γ) together exacerbates EV71-induced pulmonary
EV71 Proteins Attenuate IFN-γ Signaling 6
106 abnormality with edema in mice (39). Infection by EV71 may alter IFN-γ responses, leading
107 to escape from both antiviral strategies and immunopathogenesis. The present study was
108 therefore designed to examine the role of IFN-γ in EV 71 infection and to determine how
109 EV71 amends IFN-γ signaling.
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EV71 Proteins Attenuate IFN-γ Signaling 7
110 MATERIALS AND METHODS
111 Cell cultures and reagents. Human muscle rhabdomyosarcoma (RD) cell line (ATCC
112 CCL-136) and murine neuroblastoma (Neuro-2a) cell line (ATCC CCL-131) were maintained
113 in media according to the instructions of the ATCC. Wild-type mouse embryonic fibroblasts
114 (MEFs), a gift from Dr. Hsiao-Sheng Liu, Department of Microbiology and Immunology,
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115 National Cheng Kung University, Taiwan, were cultured in DMEM (Invitrogen Life o
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116 Technologies) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml lo
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117 penicillin, and 50 μg/ml streptomycin in a humidified atmosphere with 5% CO2 and 95% air.
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118 The reagents and antibodies used were 3D inhibitor ATA (Sigma-Aldrich); recombinant fr
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119 mouse IFN-γ (PeproTech or R&D Systems); recombinant mouse IFN-β (R&D Systems)
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120 fluorescein isothiocyanate- or phycoerythrin-conjugated control antibodies or antibodies
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121 against mouse CD4 (clone GK 1.5; eBioscience), CD8a (clone 53-6.7; eBioscience), F4/80 i.
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122 (clone BM8; eBioscience), phospho-STAT1α/β at Tyr701 and Ser727, STAT1α/β, m
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123 phospho-Jak 2 at Tyr1007/1008, Jak2, phospho-ERK1/2 at Thr202/Tyr204, ERK1/2, g
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124 phospho-MEK1/2 at Ser217/221, MEK1/2, phospho-c-Raf at Ser338, c-Raf (Cell Signaling n

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125 Technology), V5 tag, IFN-γR1, IFN-γR2 (Abcam), or β-actin (Chemicon International); and
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126 Alexa Fluor 488- and horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit, ,

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127 and donkey anti-goat IgG (Invitrogen Life Technologies). 1
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128 The virus and mice. EV71 strain M2 was propagated and titrated on RD cell monolayers
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129 and was used to infect mice as previously described (40). C57BL6/J mice and
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130 C57BL6/J-derived mice deficient in IFN-γR1 (B6.129S7-Ifngr1tm1Agt/J) were purchased from
131 The Jackson Laboratory and maintained under specific pathogen-free conditions in the
132 Laboratory Animal Center of our college.
133 Infection of mice. Animal work in this study was carried out in strict accordance with the
134 recommendations in the Guide for the Care and Use of Laboratory Animals of the National
EV71 Proteins Attenuate IFN-γ Signaling 8
135 Institutes of Health, the Animal Welfare Act and US federal law. All mouse experiment
136 protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of
137 National Cheng Kung University (IACUC Approval No.: 98012). Fourteen-day-old mice
138 were mock-infected or infected with 3 × 105 PFU per mouse of EV71 strain M2 by
139 intraperitoneal injection and monitored for survival rate and disease progression for 30 days.
140 The disease severity of infected mice was graded from 0 to 5 as follows: 0, health; 1, ruffled D
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141 hair; 2, weakness in hind limbs; 3, paralysis in single hind limb; 4, paralysis in both hind n
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142 limbs; and 5, death. In separate experiments, mice were anaesthetized, and mouse blood was
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143 collected. After perfusion with ice-cold phosphate-buffered saline (PBS) by intracardiac
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144 injection, mouse tissues were harvested, frozen, homogenized, frozen, and sonicated. The m

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145 resulting samples were assayed for viral titers by plaque assay using RD cell monolayers. tt
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146 IFN-γ measurement. Mouse blood was processed into sera. Mouse brains were frozen and i.
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147 homogenized in ice-cold PBS containing a protease inhibitor cocktail (Sigma-Aldrich). The m
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148 brain homogenates were centrifuged to obtain supernatants. The supernatants and sera were g
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149 subjected to enzyme-linked immuno-sorbent assay to measure mouse IFN-γ (R&D Systems) n

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150 using the commercially available kit according to the manufacturer’s instructions.
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151 Flow cytometry. Spleens and brains were harvested from mice perfused with PBS at day 9 2
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152 post-infection, and leukocytes were isolated from the organs and stained with antibodies 9

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153 specific for mouse leukocytes or control antibodies as previously described (41). To detect the
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154 expression of viral proteins and IFN-γ receptors, cells were stained with antibodies against V5
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155 tag, IFN-γR1, or IFN-γR2 and then incubated with a mixture of Alexa Fluor 488-conjugated
156 goat anti-rabbit IgG. Stained cells were analyzed using flow cytometry (FACSCalibur; BD
157 Biosciences) with excitation set at 488 nm; emission was detected with the FL-1 (515-545 nm)
158 and FL-2 (564-606 nm) channels. Samples were analyzed using CellQuest Pro 4.0.2 software
159 (BD Biosciences), and quantification was performed using WinMDI 2.8 software (The
EV71 Proteins Attenuate IFN-γ Signaling 9
160 Scripps Institute). Small cell debris was excluded by gating on a forward scatter plot.
161 IFNs treatment. Neuro-2a cells were pre-treated with 25 or 100 units/ml of recombinant
162 mouse IFN-β or IFN-γ for 24 h and infected with EV71 strain M2. Cultures were collected 24
163 and 36 h post-infection to determine viral titers by plaque assay.
164 Plasmid construction and transfection. To construct plasmids expressing each of the
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165 EV71 proteins, the full-length cDNAs encoding EV71 VP4, VP3, VP2, VP1, 2A, 2B, 2C, 3A, w
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166 3B, 3C, or 3D protein were amplified from an EV71/MP4 infectious clone, pEV71/MP4/y-5, lo
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167 by PCR with the corresponding viral gene-specific primer pairs (32, 42). PCR products were d

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168 cloned into the pcDNA3.1/V5-His-TOPO vector using a pcDNA3.1/V5-His TOPO TA o
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169 Expression Kit (Invitrogen) according to the manufacturer's instructions. Viral protein h
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170 expression plasmids and a control plasmid, pcDNA3.1(−), were isolated and purified using an //
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171 EndoFree Plasmid Maxi Kit (Qiagen) to eliminate endotoxin contamination. MEFs were a
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172 transfected with the plasmids using Lipofectamine 2000 (Invitrogen Life Technologies) and .
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173 cultured for 3 days. Stable cell lines were selected by culturing transfected cells in medium /

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174 containing 1.8-2.0 µg/ml of geneticin (Invitrogen Life Technologies) for 14 days. The A
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175 expression of viral genes and proteins in stable cell lines were confirmed using RT-PCR with il
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176 the gene-specific primer pairs (32, 42) and immunoblot with V5 tag, respectively. For STAT1 2
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177 transfection, transient transfection was performed using an MP-100 Microporator (Digital 9

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178 Biotechnology, Korea) according to the manufacturer’s instructions for optimization and
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179 usage. The pSG5 construct expressing STAT1 was provided by Chien-Kuo Lee (National
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180 Taiwan University).
181 RT-PCR analysis. Total RNA was extracted from mouse brain stems or
182 plasmid-transfected cells using Trizol reagent (Invitrogen Life Technologies), and 5 µg of
183 total RNA was converted to cDNA using Moloney murine leukemia virus reverse
184 transcriptase (Promega). PCR amplification was performed on 4 µl of cDNA sample.
EV71 Proteins Attenuate IFN-γ Signaling 10
185 Numbers of amplification cycles and annealing temperatures were optimized for the specific
186 primer pairs for viral genes and mouse IFN-γ and β-actin cDNA sequences (32, 42, 43). PCR
187 products were electrophoresed on 1.2% agarose gels. The gels were analyzed, and the
188 intensity of IFN-γ band was quantified using ImageJ software and normalized to the intensity
189 of β-actin band in the sample.
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190 Cytotoxicity assay. To evaluate cell damage, the activity of lactate dehydrogenase released o
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191 in culture supernatants was assayed using a colorimetric assay with Cytotoxicity Detection kit lo
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192 (Roche Diagnostics, UK) according to the manufacturer’s instructions. A microplate reader
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193 (SpectraMax 340PC; Molecular Devices) was used to measure the absorbance at 620 nm with fr
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194 a reference wavelength of 450 nm, and data were analyzed with Softmax Pro software
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195 (Molecular Devices).
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196 Luciferase reporter assay. For the luciferase reporter assay, the cells were transiently a
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197 co-transfected with the IRF1 promoter-driven firefly luciferase reporter (0.2 μg) and 0.01 μg .
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198 of Renilla luciferase-expressing plasmid (pRL-TK; Promega) using the GeneJammer /

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199 transfection reagent (Stratagene). Twenty-four h after the transfection, the cells were treated A
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200 with IFN-γ for 0.5 h, lysed, and then harvested to measure firefly and Renilla luciferase il
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201 activities using a luciferase assay system (Dual-Glo; Promega). For each lysate, the firefly 2
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202 luciferase activity was calculated as relative light unit (RLU). 9

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203 Western blotting. Both procedures were described previously (44). In brief, total cell g
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204 lysates were extracted, and proteins were separated using SDS-polyacrylamide gel t
205 electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore
206 Corporation). After blocking with 5% bovine serum albumin (Sigma-Aldrich), blots were
207 incubated with the indicated antibodies and developed using an ECL Western blot detection
208 kit (Millipore Corporation) according to the manufacturer’s instructions. The relative signal
209 intensity was quantified using ImageJ software (version 1.41o) from W. Rasband (National