Sequence-specific modifications enhance the broad spectrum antiviral response activated by RIG-I ...

35 Pages·2015·1.14 MB·English

JVI Accepted Manuscript Posted Online 27 May 2015
J. Virol. doi:10.1128/JVI.00845-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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8 Sequence-specific modifications enhance the broad spectrum antiviral response
9 activated by RIG-I agonists D
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14 Cindy Chiang1, Vladimir Beljanski1, Kevin Yin1, David Olagnier1,3, Fethia Ben Yebdri3, e
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15 Courtney Steel1, Marie-Line Goulet3, Victor R. DeFilippis2, Daniel N. Streblow2, Elias K. f
16 Haddad1, Lydie Trautmann1, Ted Ross1, Rongtuan Lin3 and John Hiscott1# ro
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21 1Vaccine & Gene Therapy Institute of Florida, Port St. Lucie, FL i.
22 2Vaccine & Gene Therapy Institute – Oregon Health and Science University, Beaverton, as
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24 3Lady Davis Institute - Jewish General Hospital, McGill University, Montreal, CA o
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29 #Correspondence should be addressed to: u
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31 Dr. John Hiscott 2
32 VGTI Florida 1
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33 9801 Discovery Way 2
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34 Port St. Lucie FL 34987 1
35 Email: [email protected] 9

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38 Running title: Sequence-specific RIG-I agonists inhibit RNA viruses s
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39
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40 ABSTRACT
41 The cytosolic RIG-I receptor plays a pivotal role in the initiation of the immune response against
42 RNA virus infection by recognizing short 5’-triphosphate (5’ppp) containing viral RNA and
43 activating the host antiviral innate response. In the present study, we generated novel 5’ppp
44 RIG-I agonists of varied length, structure, and sequence and evaluated the generation of the
45 antiviral and inflammatory response in human epithelial A549 cells, human innate immune
46 primary cells, and murine models of influenza and chikunguna viral pathogenesis. A 99 D
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47 nucleotide, uridine-rich, hairpin 5’-pppRNA termed M8 stimulated an extensive and robust w
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48 interferon response compared to other modified 5’pppRNA structures, RIG-I aptamers, or poly lo
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49 (I:C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate d
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50 antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I, and
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51 independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 o
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52 effectively inhibited influenza and dengue viral replication in vitro. Furthermore, multiple strains h
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53 of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for p
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54 influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo jv
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55 prolonged survival and reduced lung viral titers of mice challenged with influenza, as well as a
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56 reduced chikungunya-associated foot swelling and viral load. Altogether, these results
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57 demonstrate that 5’pppRNA can be rationally designed to achieve a maximal RIG-I-mediated r
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58 protective antiviral response against human pathogenic RNA viruses. o
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59 J
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60 IMPORTANCE n
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61 The development of novel therapeutics to treat human pathogenic RNA viral infections is an r
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62 important goal that seeks to reduce spread of infection and to improve human health and safety. 2
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63 This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory ,
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64 properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, 1
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65 uridine-rich RIG-I agonist generated a protective antiviral response in vitro and in vivo and was b
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66 effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, g
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67 highlighting the robust activity and potential clinical use of the 5’pppRNA against RNA virus e
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68 infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an t
69 attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing
70 issue in which a need for new and more effective options persists.
71
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72 INTRODUCTION
73 Human pathogenic RNA viral infections, including influenza, dengue, and chikungunya, pose
74 significant threats to human health and safety. For this reason, the development of prophylactic
75 and therapeutic antivirals to treat and limit spread of infection remains a growing unmet medical
76 need. Currently, there are no therapeutics for the prevention or treatment of dengue or
77 chikungunya infections, and approved antiviral compounds to treat influenza have significant
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78 problems associated with their use. For instance, anti-influenza agents such as amantadine and
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79 rimantadine block virus uncoating, but are not recommended for currently circulating influenza A n
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80 or B strains because of widespread resistance (1). Oseltamivir, a neuraminidase inhibitor, is a
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81 also active against influenza A and B at early stages of infection but has generated drug- e
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82 resistant mutants (2, 3). Therapies that harness and activate the natural immune defense may f
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83 circumvent the issues of the emergence of drug resistance and off-target effects. m

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84
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85 The innate immune system provides the initial barrier against viral infection, initiating a cascade :
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86 of signaling pathways and sensors that detect and clear the intruding virus. RNA viruses jv
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87 possess pathogen associated molecular patterns (PAMPs) which are sensed by pattern s
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88 recognition receptors (PRR) (4-8). Toll-like receptor (TLR) and RIG-I-like receptor (RLR) .
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89 families generate an innate immune response upon recognition of broadly conserved PAMPs on g
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90 viruses and bacteria(9). RIG-I recognizes short dsRNA oligonucleotides of <100 nucleotides in n

91 length bearing 5'-triphosphate or 5’-diphosphate termini (10), while MDA5 generally recognize Ja
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92 longer, dsRNA (>300 nucleotides) lacking a 5’-triphopshate moiety. RIG-I detects viral RNA u
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93 through its helicase domain (11-14) leading to conformational changes that expose the effector ry

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94 caspase activation and recruitment domain (CARD) which in turn interacts with the 1
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95 mitochondrial adaptor MAVS(15-17). MAVS serves as a signaling platform for protein 2
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96 complexes that trigger activation of the transcription factors NF-κB and IFN regulatory factors 1
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97 (IRF)-3 and -7 leading to the induction of antiviral programs that include production of type I IFN, b
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98 as well as pro-inflammatory cytokines and antiviral factors (18-23). A secondary response is g
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99 induced by IFN binding to the type I IFN receptors (IFNα/βR), which activate the JAK-STAT s
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100 pathway, and induce interferon stimulated genes (ISGs) and the antiviral immune response (24,
101 25). More recently, RIG-I has been shown to function both as an innate sensor and an antiviral
102 factor by triggering downstream interferon signaling events and disrupting the interaction
103 between hepatitis B virus (HBV) polymerase and pgRNA(26). Overall, novel therapies
104 specifically targeting the retinoic acid-inducible gene-I (RIG-I) pathway have the potential to
105 elicit a broad-spectrum, antiviral, inflammatory and immune modulatory response and thus
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106 represent an attractive strategy for the design and development of novel and improved antiviral
107 therapies.
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109 We previously reported the antiviral activity of a short in vitro-synthesized 5’-triphosphate RNA
110 (5′pppRNA) derived from the 5′ and 3′ untranslated regions (UTRs) of the VSV genome (termed
111 “WT” 5’pppRNA) (27). Pre-treatment with WT 5’pppRNA activated the RIG-I signaling pathway
112 and triggered a robust antiviral response that significantly decreased infection by several D
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113 pathogenic viruses, including dengue, HCV, H1N1 influenza A/PR/8/34 and HIV-1. In vivo, w
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114 intravenous delivery of the WT 5’pppRNA stimulated an antiviral state that inhibited a broad lo
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115 spectrum of RNA viruses and protected mice from lethal influenza virus challenge (27, 28). d
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117 The nature of the ligand recognized by RIG-I has been the subject of numerous studies. o
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118 Structural motifs, length, and sequence of virus-derived 5’pppRNA and other RNA agonists h
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119 have been analyzed and found to play critical roles in the response to viral infection. In vitro p
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120 synthesized RNA with 5′ terminal triphosphate moiety was first identified as a RIG-I agonist (29, jv
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121 30); however more recently, it was discovered that a 5’-diphosphate termini could also be a
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122 recognized be RIG-I to mediate an antiviral response (10). RIG-I stimulation was dependent on
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123 double stranded RNA at least 20 nucleotides long possessing blunt base pairing at the 5′ end r
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124 (30, 31). Interestingly, ligands recognized by RIG-I include double stranded RNA, found in o
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125 conserved 5′ and 3′ UTRs of negative single strand RNA virus genomes, displaying high base J
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126 pair complementarity and a panhandle structure (31). n
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127 r
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128 Most studies on RIG-I antiviral properties have used virus-derived 5’pppRNA or defective 2
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129 interfering particles, or commercially available synthetic 5’pppRNA to trigger the RIG-I antiviral ,
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130 response. In the present study, we report for the first time the sequence-specific activity of a 1
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131 novel RIG-I agonist active against a number of RNA viruses both in vitro and in vivo. b
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132 Modifications to the structure, length, and sequence of a prototypical 5’triphosphate containing g
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133 RNA significantly potentiated the host antiviral response against influenza, dengue and e
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134 chikungunya virus infections while maintaining the specificity for interaction with the RIG-I t
135 cytosolic sensor.
136
137 MATERIALS AND METHODS
138 In vitro transcription and gel analysis. RIG-I agonists were synthesized by designing
139 complementary forward (F) and reverse (R) primers with a T7 promoter (Integrated DNA
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140 Technologies) as follows: VSV WT F, 5’-
141 gacgaagacaaacaaaccattattatcattaaaattttattttttatctggttttgtggtcttcgtctatagtgagtcgtattaatttc-3’; R, 5’-
142 gaaattaatacgactcactatagacgaagaccacaaaaccagataaaaaataaaattttaatgataataatggtttgtttgtcttcgtc-
143 3’; M1 F, 5’- gacgaagaccacaaaaccattattatcattaaaattttattttttatctggttttgtggtcttcgtctatagtgagtcgtatta-
144 atttc-3’; R, 5’- gaaattaatacgactcactatagacgaagaccacaaaaccagataaaaaataaaattttaatgataataatgg-
145 ttttgtggtcttcgtc-3’; M2 F, 5’- gacgaagacaaacaaaccagataaaaaattaaaattttattttttatctggttttgtggtcttcgtc-
146 tatagtgagtcgtattaatttc-3’; R, 5’-gaaattaatacgactcactatagacgaagaccacaaaaccagataaaaaataaaattt- D
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147 taattttttatctggtttgtttgtcttcgtc -3’; M3 F, 5’- gactccgacaaacaaaccattattatcattaaaattttattttttatctggttttgt- w
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148 ggtcttcgtctatagtgagtcgtattaatttc -3’; R, 5’- gaaattaatacgactcactatagacgaagaccacaaaaccagataaa- lo
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149 aaataaaattttaatgataataatggtttgtttgtcggagtc-3’; M4 F, 5’-gacgaagaccacaaaaccattatggttttgtggtcttc- d
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150 gtctatagtgagtcgtattaatttc -3’; R, 5’-gaaattaatacgactcactatagacgaagaccacaaaaccataatggttttgtggtc-
f
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151 ttcgtc-3’; M5 F, 5’-gacgaagaccacaaaaccagataaaaaattattttttatctggttttgtggtcttcgtctatagtgagtcgtatta- o
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152 atttc-3’; R, 5’-gaaattaatacgactcactatagacgaagaccacaaaaccagataaaaaataattttttatctggttttgtggtcttc- h
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153 gtc-3’; M6 F, 5’- gacgaagaccacaaaaccagataaaaaaaaaaattatttttttttttatctggttttgtggtcttcgtctatagtga- p
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154 gtcgtattaatttc-3’; R, 5’- gaaattaatacgactcactatagacgaagaccacaaaaccagataaaaaaaaaaataattttttttt- jv
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155 ttatctggttttgtggtcttcgtc -3’; M7 F, 5’- gacgaagaccacaaaaccagataaaaaaaaaaaaaaaattatttttttttttttttt- a
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156 atctggttttgtggtcttcgtctatagtgagtcgtattaatttc-3’; R, 5’-gaaattaatacgactcactatagacgaagaccacaaaac-
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157 cagataaaaaaaaaaaaaaaataattttttttttttttttatctggttttgtggtcttcgtc-3’; M8 F, 5’- gaaattaatacgactcactat- r
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158 agacgaagaccacaaaaccagataaaaaaaaaaaaaaaataattttttttttttttttatctggttttgtggtcttcgtc-3’; R, 5’- gaa- o
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159 attaatacgactcactatagacgaagaccacaaaaccagataaaaaaaaaaaaaaaaaaaaaaaaaataattttttttttttttttttttttt J
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160 tttatctggttttgtggtcttcgtc-3’; M8A F, 5’- gacgaagaccacaaaaccagataataataataataataataataataattatt- n
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161 attattattattattattattattatctggttttgtggtcttcgtctatagtgagtcgtattaatttc-3’; R, 5’- gaaattaatacgactcactata- r
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162 gacgaagaccacaaaaccagataataataataataataataataataataattattattattattattattattattatctggttttgtggtcttc 2
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163 gtc-3’; M8B F, 5’-gcatcgtacagctgcagttacttaattcattgaatatcttatgcatgtagtacatgcataagatattcaatgaatt- ,
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164 aagtaactgcagctgtacgatgctatagtgagtcgtattaatttc-3’; R, 5’- gaaattaatacgactcactatagcatcgtacagctg- 1
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165 cagttacttaattcattgaatatcttatgcatgtactacatgcataagatattcaatgaattaagtaactgcagctgtacgatgc-3’; M8C b
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166 F, 5’- gacgaagaccacaaaaccagatcacacacacacacacacacacacacattatgtgtgtgtgtgtgtgtgtgtgtgtgatctg- g
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167 gttttgtggtcttcgtctatagtgagtcgtattaatttc-3’; R, 5’- gaaattaatacgactcactatagacgaagaccacaaaaccaga- e
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168 tcacacacacacacacacacacacacataatgtgtgtgtgtgtgtgtgtgtgtgtgatctggttttgtggtcttcgtc-3’; M8D F, 5’- t
169 gaccaaccagacaacaagagataaaaaaaaaaaaaaaaaaaaaaaaaattattttttttttttttttttttttttttatctcttgttgtctggttg-
170 gtctatagtgagtcgtattaatttc-3’; R, 5’- gaaattaatacgactcactatagaccaaccagacaacaagagataaaaaaaaa-
171 aaaaaaaaaaaaaaaaataattttttttttttttttttttttttttatctcttgttgtctggttggtc-3’. Primers were annealed then
172 synthesized with an in vitro transcription kit (Ambion) for 16 hours. RNA transcripts were DNase
173 digested for 15 minutes at 37°C then purified with an miRNeasy kit (Qiagen). RNA was
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174 analyzed on a denaturing 15% TBE-urea polyacrylamide gel (Bio-rad) following digestion with
175 50 ng/ul of RNase A (Ambion) or 100mU/ul of DNase I (Ambion) for 30 minutes. Control wild
176 type RNA is the dephosphorylated form of the WT sequence purchased from IDT. Secondary
177 structure was predicted using the RNAfold WebServer (University of Vienna).
178
179 Cell culture, transfections, and luciferase assays. Lung epithelial A549 cells were grown in
180 F12K (ATCC) supplemented with 10% FBS (Access Cell Culture). Transfection of RNA and D
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181 siRNA in A549 cells were performed with Lipofectamine RNAiMax (Invitrogen) for 18-24 hours w
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182 and 48 hours, respectively. Transfection of RNA and siRNA in Mo-DC were performed with lo
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183 HiPerFect transfection reagent (Qiagen). Poly (I:C) LMW was purchased from Invivogen. For d
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184 siRNA knockdown, A549 cells were transfected with 30pmol of control siRNA (sc-37007),
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185 human RIG-I (sc-61480), TLR3 (sc-36685), MDA5 (sc-61010), TLR7 (sc-40266) or TLR8 (sc- m
186 40268) (Santa Cruz Biotechnologies) using Lipofectamine RNAiMax according to the h
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187 manufacturer’s guidelines. For luciferase assay, 200ng IFN-β/pGL3 and 100ng pRL-TK p
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188 plasmids were co-transfected with 5’pppRNA using Lipofectamine RNAiMax for 24h. Reporter jv
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189 gene activity was measured by Dual-Luciferase Reporter Assay (Promega) according to the a
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190 manufacturer’s instructions. Relative luciferase activity was measured as fold induction.
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191 Oseltamivir was purchased from AvaChem Scientific. rg
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192 o
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193 Monocyte isolation and differentiation into monocyte-derived dendritic cells. Human J
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194 peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy, u
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195 seronegative volunteers in a study approved by the IRB and by the VGTI-FL Institutional r
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196 Biosafety Committee (2011-6-JH1). Written informed consent approved by the VGTI-FL Inc. 2
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197 ethics review board (FWA#161) was provided to study participants. Research conformed to 2
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198 ethical guidelines established by the ethics committee of the OHSU VGTI and Martin Health 1
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199 System. Briefly, PBMC were isolated from freshly collected blood using the Ficoll-Paque™ b
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200 PLUS medium (GE Healthcare Bio) as per manufacturer’s instructions. CD14+ monocytes were g
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201 isolated by positive selection using CD14 microbeads and a magnetic cells separator as per kit e
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202 instructions (Miltenyi Biotech). Purified CD14+ monocytes were cultured for 7 days in 100 mm
203 dishes (15x106 cells) in 10 mL of complete monocyte differentiation medium (Miltenyi Biotech).
204 On day 3, the medium was replenished with fresh medium. Differentiation was confirmed by
205 flow cytometry analysis on day 7. CD14low/CD1ahigh/DC-SIGNhigh cells were used for
206 experiments.
207
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208 Quantitative real-time RT-PCR. Total RNA was isolated from cells using RNeasy kit (Qiagen)
209 according to the manufacturer’s instructions. RNA was reverse transcribed using the
210 SuperScript® VILO cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions.
211 PCR primers were designed using Roche’s Universal Probe Library Assay Design Center
212 (www.universalprobelibrary.com) and purchased from Integrated DNA Technology (USA).
213 Quantitative RT-PCR was performed on a LightCycler® 480 Probes Master (Roche.) All data
214 are presented as a relative quantification with efficiency correction based on the relative D
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215 expression of target gene versus GAPDH as the invariant control. w
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216 lo
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217 Fluidigm BioMark assay. The 5’pppRNA BioMark experiment was performed with Mo-DC d
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218 derived from 3 independent healthy donors. Total RNA and cDNA were prepared as described
f
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219 above. cDNA along with the entire pool of primers were pre-amplified for 18 cycles using o
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220 TaqMan PreAmp Master Mix as per manufacturer’s protocol (Applied Biosystems). Pre-amped h
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221 cDNA was treated with Exonuclease I (New England Biolabs) then combined with 2X FastStart p
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222 TaqMan Probe MasterRoche), GE sample loading buffer (Fluidigm) and Taq Polymerase jv
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223 (Invitrogen). Assays were prepared with 2X assay loading reagent (Fluidigm), primers (IDT) and a
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224 probes (Roche). Samples and assays were loaded in their appropriate inlets on a 48.48
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225 BioMark chip. The chip was run on the BioMark HD System (Fluidigm) for 40 cycles. Raw Ct r
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226 values were calculated by the real time PCR analysis software (Fluidigm) and software- o
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227 designated failed reactions were discarded from analysis. All data are presented as a relative J
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228 quantification with efficiency correction based on the relative expression of target gene versus n
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229 the geomean of (GAPDH+Actin+b2 microglobulin) as the invariant control. The N-fold r
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230 differential expression of mRNA gene samples was expressed as 22DDCt. The heatmaps were 2
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231 produced with the following package; pheatmap: Pretty Heatmaps. R package version 0.7.7 ,
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232 http://CRAN.R-project.org/package=pheatmap. Gene level expression is shown as 2DDCt or 1
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233 gene-wise standardized expression (Z score). The sequences of forward (F) and reverse (R) b
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234 primers used as well as their complementary probes are as follows: BIRC3 (probe 44) F, 5’- g
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235 gactgggcttgtccttgct-3’; R, 5’-aagaagtcgttttcctcctttgt-3’; CCL3 (probe 74) F, 5’- e
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236 tgctcagaatcatgcaggtc-3’; R, 5’-gcgtgtcagcagcaagtg-3’; CCL5 (probe 59) F, 5’- t
237 tgcccacatcaaggagtattt-3; R, 5’-tttcgggtgacaaagacga-3; CXCL10 (probe 34) F, 5’-
238 gaaagcagttagcaaggaaaggt-3; R, 5’-gacatatactccatgtagggaagtga-3; DDX58 (probe 6) F, 5’-
239 tgtgggcaatgtcatcaaaa-3; R, 5’-gaagcacttgctacctcttgc-3; DENV2 (probe 5) F, 5’-
240 atcctcctatggtacgcacaaa-3; R, 5’-ctccagtattattgaagctgctatcc-3; GAPDH (probe 60) F, 5’-
241 agccacatcgctcagacac-3; R, 5’-gcccaatacgaccaaatcc-3; IFIT1 (probe 50) F, 5’-
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242 gcctaatttacagcaaccatga-3; R, 5’-gcctaatttacagcaaccatga-3; IFIT2 (probe35) F, 5’-
243 atataggtctcttcagcatttattggt-3; R, 5’-caaggaattcttattgttctcactca-3; 5’--3; IFITM1 (probe 60) F, 5’-
244 cacgcagaaaaccacacttc-3; R, 5’-tgttcctccttgtgcatcttc-3; IFITM2 (probe 75) F, 5’-
245 tgaaccacattgtgcaaacc-3; R, 5’-ctcctccttgagcatctcgt-3; IFITM3 (probe 76) F, 5’-
246 agatgctcaaggaggagcac-3; R, 5’-gatgtggatcacggtggac-3; IFNAR1 (probe 65) F, 5’-
247 atttacaccatttcgcaaagc-3; R, 5’-cactattgccttatcttcagcttcta-3; IFNAR2 (probe 87) F, 5’-
248 tagcctccccaaagtcttga-3; R, 5’-aaatgacctccaccatatcca-3; IFNB1 (probe 20) F, 5’- D
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249 ctttgctattttcagacaagattca-3; R, 5’-gccaggaggttctcaacaat-3; IL1A (probe 66) F, 5’- w
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250 tgacgccctcaatcaaagta-3; R, 5’-tgacttataagcacccatgtcaa-3; IL1B (probe 78) F, 5’- lo
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251 tacctgtcctgcgtgttgaa-3; R, 5’-tctttgggtaatttttgggatct-3; IL6 (probe 40) F, 5’- d
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252 gatgagtacaaaagtcctgatcca-3; R, 5’-ctgcagccactggttctgt-3; IL8 (probe 72) F, 5’-
f
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253 agacagcagagcacacaagc-3; R, 5’-tggttccttccggtggt-3; IL10 (probe 65) F, 5’- o
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254 gctggacaacttgttgttaaagg-3; R, 5’-ctcagacaaggcttggcaac-3; IL12A (probe 50) F, 5’- h
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255 cactcccaaaacctgctgag-3; R, 5’-tctcttcagaagtgcaagggta-3; IL28RA (probe 12) F, 5’- p
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256 cccccactggatctgaagta-3; R, 5’-gagtgactggaaatagggtcttg-3; IL29 (probe 75) F, 5’- jv
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257 cctgaggcttctccaggtg-3; R, 5’-ccaggaccttcagcgtca -3; IRF3 (probe 18) F, 5’- cttggaagcacggcctac - a
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258 3; R, 5’-cgggaacatatgcaccagt-3; IRF7 (probe 72) F, 5’-agctgtgctggcgagaag-3; R, 5’-
.
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259 ttggagtccagcatgtgtg-3; ISG15 (probe 23) F, 5’-gcgaactcatctttgccagta-3; R, 5’- r
g
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260 ccagcatcttcaccgtcag-3; MX1 (probe 79) F, 5’-ttcagcacctgatggccta-3; R, 5’- o
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261 aaagggatgtggctggagat-3; MX2 (probe 9) F, 5’-cagacctgaccatcattgacc-3; R, 5’- J
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262 tgatgagagccttgatctgc-3; SOCS3 (probe 55) F, 5’-gacctgaagggaaccatcct-3; R, 5’- n
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263 tgtgttttcggtgactgtcc-3; STAT1 (probe 74) F, 5’-ggatcagctgcagaactggt-3; R, 5’- r
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264 tttctgttccaattcctccaa-3; TANK (probe 80) F, 5’-gaggaatagtctacaaaggaagacttg-3; R, 5’- 2
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265 actataaaggatggagtaaatgacagg-3; TLR3 (probe 22) F, 5’-aaggctagcagtcatccaaca-3; R, 5’- ,
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266 agcaacttcatggctaacagtg-3; TLR7 (probe 5) F, 5’-ccagtgtctaaagaacctggaaac-3; R, 5’- 1
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267 tcagggacagtggtcagttg-3; TNF (probe 79) F, 5’-gacaagcctgtagcccatgt-3; R, 5’-tctcagctccacgccatt- b
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268 3. g
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269 e
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270 Immunoblot analyses. Whole cell extracts were separated in 4-20% acrylamide Mini-Protean t
271 ® TGX precast gels (Bio-rad) by SDS-PAGE and transferred to an Immobilon-PSQ PVDF
272 membrane (Millipore) for 1 hour at 100V in a buffer containing 30mM Tris, 200mM glycine and
273 20% methanol. Membranes were blocked for 1 hour at room temperature in blocking buffer
274 (Odyssey) then probed with the following primary antibodies: anti-RIG-I (EMD Millipore), anti-
275 IFIT1 (Thermo Fisher Scientific), anti-ISG56 (Cell Signaling), anti-STAT1 (Cell Signaling), anti-
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276 pIRF3 S396 (Cell Signaling), anti-IRF3 (Cell Signaling), anti-TLR3 (Cell Signaling), anti-MDA5
277 (Cell Signaling), anti-β-actin (Odyssey), anti-DenV2 E protein (Santa Cruz Biotechnology) or
278 anti-NS1 (Santa Cruz Biotechnology). Antibody signals were detected by immunofluorescence
279 using the IRDye® 800CW and IRDye ® 680RD secondary antibodies (Odyssey) and the LI-
280 COR imager (Odyssey.)
281
282 Flow cytometry analysis. Percentage of dengue-infected cells was determined by standard D
o
283 intracellular staining (ICS) using a mouse IgG2a mAb, specific for dengue E protein (clone 4G2) w
n
284 followed by staining with a secondary anti-mouse antibody coupled to PE (Biolegend). pSTAT1 lo
a
285 geomean fluorescence was determined by PhosFlow staining as previously reported (32) with d
e
d
286 pSTAT1 Y701 Pacific Blue antibody (BD Biosciences). Cells were analyzed on an LSRII flow
f
r
o
287 cytometer (Becton Dickinson). Calculations and population analyses were done using FACS m
288 Diva software. h
t
t
289 p
:
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290 Virus production and in vitro infection. Dengue serotype 2 strain New Guinea C (NGC) was jv
i.
a
291 used to infect confluent monolayers of C6/36 insect cells at an MOI of 0.5. Virus was allowed to s
m
292 adsorb for 1 hour at 28°C in serum-free DMEM. Serum-free DMEM was used to wash the
.
o
293 monolayer then replaced with DMEM/2% FBS. After 7 days of infection, the medium was rg
/

294 harvested, cleared by centrifugation (1100g, 10min), and the supernatant was concentrated by o
n

295 centrifugation (1100g) through a 15ml Amicon° Centrifugal Filter Unit (Millipore). The virus was J
a
n
296 concentrated by ultracentrifugation on a sucrose density gradient (20% sucrose cushion) using u
a
297 a Sorvall WX 100 Ultracentrifuge (ThermoScientific) for 2 hours at 134,000g, 10°C and the r
y

298 brake turned off. Concentrated virus was then washed to remove sucrose using a 15ml 2
1
,
299 Amicon® tube. After 2 washes, the virus was resuspended in DMEM/0.1% BSA. Titers of 2
0
300 dengue stocks were determined by FACS, after infecting Vero, immunofluorescence staining of 1
9
301 intracellular dengue E protein 24 hours post infection. For dengue challenge experiments, A549 b
y
302 cells and Mo-DCs were infected using dengue at an MOI of 0.5 in serum-free medium for 1 hour g
u
303 at 37°C. Medium was replaced with complete medium for 24h prior to analysis. e
s
t
304 For in vitro influenza challenge experiments, A549 cells and Mo-DCs were infected with various
305 influenza strains (MOI 0.2 or 2) in a small volume of serum-free medium for 1 hour at 37°C.
306 Medium was replaced with complete medium for 24h prior to analysis.
307 Influenza reassortant H5N1 virus (H5N1-PR8) was generated using hemagglutinin (HA) and
308 neuraminidase (NA) genes were derived from the H5N1 virus (HA from influenza
309 A/Vietnam/1203/2004 and NA from influenza A/Thailand/1(KAN-1)/2004). The internal viral
9
310 proteins were derived from the A/Puerto Rico/8/1934 (PR8) mouse adapted influenza A virus.
311 The propagation of the H5N1-PR8 reassortant viruses was performed using MDCK cells. All
312 procedures with live dengue and influenza were performed in a biosafety level 2+ facility at the
313 Vaccine and Gene Therapy Institute-Florida.
314
315 Plaque assay. Plaque assay was performed on supernatants from influenza-infected A549
316 cells. MDCK cells in 6-well plates were grown to confluency and washed twice with DMEM D
o
317 containing 1% Pen/Strep (Life Technologies). Serial dilutions of virus (1:10) were inoculated on w
n
318 MDCK cells in a volume of 100μl and adsorbed for 1 hour at room temperature, rocking every lo
a
319 15 minutes. Wells were washed twice with DMEM containing antibiotics. 2x Leibovitz’s L-15 d
e
d
320 medium (w/L-glutamine) (Cambrex) was prepared with HEPES, 7.5% sodium bicarbonate,
f
r
321 gentamicin, and TPCK-trypsin (0.6mg/ml) (Sigma-Aldrich) then combined with 1.6% agarose o
m
322 and overlaid on infected-cells. Plates were incubated at 37°C and monitored daily for plaques. h
t
t
323 48 hours post-infection, plaques were stained with 1% crystal violet/20% ethanol, counted, and p
:
/
/
324 viral titers were calculated. jv
i.
325 a
s
m
326 In vivo administration of 5’pppRNA and viral infection. BALB/c mice (6-8 weeks of age,
.
o
327 Jackson Laboratories) were housed in cage units, fed ad libitum, and cared for under USDA rg
/

328 guidelines for laboratory animals. Prior to 5'pppRNA injections and viral challenge, mice were o
n
329 anesthetized with IsoSol (Patterson Veterinary) and 5 μg of purified 5'pppRNA was injected IV J
a
330 via tail vein. The 5’pppRNA was complexed with in vivo-jetPEI (PolyPlus, France) at an N/P n
u
a
331 ratio of 8 according to the manufacturer instructions. Mice were then challenged with H5N1-RE r
y

332 (5,000 pfu in 50 μL of PBS). Animals were monitored for survival and morbidity (weight loss, 2
1
,
333 ruffling fur, hunched back, lethargy) each day during the viral challenge. All procedures were in 2
0
334 accordance with the NRC guide for the Care and Use of Laboratory Animals and the Animal 1
9
335 Welfare Act. Lungs were isolated from mice post-mortem and snap frozen in dry ice/ethanol b
y
336 bath. Dubecco's modified Eagle medium (DMEM, 10 volumes to grams) of was then added to g
u
337 the tissue placed in a 0.7 μm cell strainer in a Petri dish and sample was muddled until it has e
s
t
338 been broken down. Remaining liquid was collected from Petri dish in sample tube for stock lung
339 homogenate sample. All procedures with influenza reassortant H5N1-PR8 were performed in a
340 biosafety level 2+ facility at the Vaccine and Gene Therapy Institute-Florida.
341 Control RNA or M8 5’pppRNA were administered to adult mice using a protocol similar to that of
342 the influenza infection in vivo model. Mice were injected intraperitoneally with 2μg RNA in
343 combination with in vivo JetPEI for 24 hours then infected with 1000 PFU chikungunya virus via
10