1 Antiviral RNA interference responses induced by Semliki Forest virus infection 1 of mosquito cells

37 Pages·2010·2.3 MB·English

JVI Accepts, published online ahead of print on 29 December 2010
J. Virol. doi:10.1128/JVI.02052-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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1 Antiviral RNA interference responses induced by Semliki Forest virus infection
2 of mosquito cells: Characterization, origin and frequency-dependent functions of
3 viRNAs.
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5 Ricky W. C. Siu, Rennos Fragkoudis, Peter Simmonds, Claire L. Donald, Margo
6 E. Chase-Topping, Gerald Barry, Ghassem Attarzadeh-Yazdi, Julio Rodriguez-
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7 Andres, Anthony A. Nash, Andres Merits1, John K. Fazakerley* and Alain Kohl* o
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9 The Roslin Institute and Centre for Infectious Diseases, University of Edinburgh, e
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10 Edinburgh EH9 1QH, Scotland, United Kingdom. o
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11 1: Institute of Technology, University of Tartu, Nooruse 1, Tartu 50411, Estonia. h
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13 *To whom correspondence should be addressed: s
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14 [email protected], [email protected] r
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15 Fax: [phone] n

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16 Phone: [phone] r
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18 Running title: RNAi-mediated inhibition of alphavirus replication 1
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20 This paper contains: Abstract, 244 words; Text, 4580 words; Figures: 9; Tables: 1. e
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21 Supplementary information: Supplementary Tables: 3; Supplementary Figures: 3.
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25
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26 Abstract
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28 RNA interference (RNAi) is an important mosquito defence mechanism against
29 arbovirus infection. In this paper we study processes underlying antiviral RNAi in
30 Aedes albopictus-derived U4.4 mosquito cells infected with Semliki Forest virus
31 (SFV) (Togaviridae; Alphavirus). Production of virus-derived small interfering RNAs
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32 (viRNAs) from viral dsRNA is a key event in this host response. dsRNA could be o
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33 formed by RNA replication intermediates, by secondary structures in RNA genomes lo
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34 or antigenomes, or both. Which of these dsRNAs is the substrate for generation of e
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35 viRNAs is a fundamental question. Here we used deep sequencing of viRNAs and o
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36 bioinformatic analysis of RNA secondary structures to gain insights into the
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37 characteristics and origins of viRNAs. An asymmetric distribution of SFV-derived /jv
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38 viRNAs with notable areas of high viRNA production (hot spots) and no/low s
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39 frequency of viRNA production (cold spots) along the length of the viral genome with r
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40 a slight bias towards production of genome-derived viRNAs over antigenome-derived n

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41 viRNAs were observed. Bioinformatic analysis suggests that hot spots of viRNA p
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42 production are rarely but not generally associated with putative secondary structures ,
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43 in the SFV genome, suggesting that most viRNAs are derived from replicative 9

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44 dsRNA. An almost identical pattern of viRNAs to those in Ae. albopictus cells was
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45 observed in Ae. aegypti-derived Aag2 cells, suggesting common mechanisms that lead s
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46 to viRNA production. Hot spot viRNAs were found to be significantly less efficient at
47 mediating antiviral RNAi than cold spot viRNAs, pointing towards a nucleic acid-
48 based viral decoy mechanism to evade the RNAi response.
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50
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51 Introduction
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53 Arboviruses are transmitted to vertebrates by arthropods such as mosquitoes or ticks
54 (58, 59). The Alphavirus genus within the Togaviridae family contains medically
55 important mosquito-borne arboviruses such as chikungunya virus and Venezuelan
56 equine encephalitis virus (42, 59). Replication in vertebrate cells of the prototype
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57 alphaviruses, Sindbis virus (SINV) and Semliki Forest virus (SFV), is well o
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58 understood (19, 23, 45). The alphavirus genome is a positive-stranded RNA with 5’ lo
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59 cap and 3’ poly-A tail. The 5’ two-thirds encode the non-structural polyprotein P1234, e
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60 which is separated into four replicase proteins nsP1-4 by proteolytic cleavage; during o
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61 replication, the genome is transcribed into a full length copy (the antigenome) which
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62 in turn serves as template for de novo transcription of viral genomes (32, 34, 57). The /jv
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63 3’ third encodes the structural polyprotein, which is translated from a subgenomic s
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64 mRNA and generates the capsid and envelope glycoproteins after proteolytic r
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65 processing (54). Alphaviruses replicate within cytoplasmic replication complexes n

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66 associated with cellular membranes, and viruses mature by budding (17, 45); p
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67 important differences between replication complex formation in vertebrate and ,
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68 mosquito cells have been described (16). Infection of mosquito cell cultures with 9

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69 alphaviruses usually starts with an acute phase characterised by efficient virus
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70 production, followed by the establishment of a persistent infection with low level s
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71 virus production (6, 10, 15).
72 The efficient control of arbovirus infection in insects such as mosquitoes is believed
73 to be due to innate immune responses, and alphaviruses such as SFV have proven to
74 be good models to study these (14). The best characterised mosquito antiviral defence
75 system to-date is RNA interference (RNAi) (14, 47), which controls arbovirus
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76 replication, spread and transmission (26, 46). Central to antiviral RNAi in insects is
77 the production of virus-derived small interfering RNAs, or viRNAs, from viral
78 double-stranded RNA (dsRNA) and assembly of an RNA-induced silencing complex
79 (RISC), followed by degradation of target single-stranded viral RNA in a sequence-
80 dependent manner (14, 25). The origin of the dsRNA substrate is not clear, and could
81 involve double-stranded regions in single viral genome or antigenome RNA
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82 molecules, or two-molecule replication intermediates (the favoured hypothesis) (38). o
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83 Mosquito orthologues of the Drosophila melanogaster RNAi proteins Dcr-2, R2D2 lo
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84 and Ago-2 are important in mosquito RNAi responses against flaviviruses and e
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85 alphaviruses (7, 8, 24, 46). In D. melanogaster, the RNAse III enzyme and DExD/H- o
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86 box RNA helicase Dcr-2 is a pattern recognition receptor (PRR) which initiates RNAi
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87 by cleavage of virus-derived long dsRNA into double-stranded viRNAs. Dcr-2 and /jv
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88 R2D2 interact during the process of viRNA generation, and integrate viRNAs into the s
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89 RISC. One of the viRNA strands (the guide strand; the passenger strand is degraded) r
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90 is retained within the RISC, which targets and recognises viral single-stranded RNA n

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91 in a sequence-specific manner and mediates cleavage through the RISC protein Ago-2 p
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92 (25). In mosquitoes infected with SINV and o’nyong-nyong virus, cloning and ,
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93 sequencing of viRNAs showed that viRNA strands are mainly 21 nucleotides (nt) in 9

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94 length. These viRNAs map asymmetrically along the complete length of the virus
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95 genome; regions generating a high frequency of viRNAs (hot spots) and regions s
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96 generating no or low frequency of viRNA (cold spots) are interspersed in an
97 apparently random manner (38, 39). However as these infections are non-
98 synchronized but reflect a mix of cells at various stages of infection, it is difficult to
99 speculate on the origins of viRNAs. It appears that arboviruses do not encode protein
100 suppressors of RNAi, unlike pathogenic insect viruses many of which encode for
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101 example dsRNA-binding proteins that counteract RNAi (14, 25). It has been
102 speculated that an RNAi-suppressive activity may exist in dengue virus-infected
103 mosquito cells (46). In addition, it is now known that there is a systemic component to
104 RNAi responses in insects (3, 44). Little is known with regard to the biogenesis of
105 arbovirus-derived viRNAs. In the case of positive-strand RNA viruses or viroids of
106 plants, the predominance of genome-derived viRNAs has led to the suggestion that
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107 secondary structures in the genome are the substrate for dicer, although replication o
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108 intermediates have also been implicated (20, 22, 37, 61). For alphaviruses, this lo
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109 remains a topic of debate, and the substrate for Dcr-2, viral dsRNA could be e
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110 replication intermediates, secondary structures, or both (14, 38). o
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111 Here we show that synchronized SFV infection of Ae. albopictus cells induced
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112 dsRNA, which mainly co-localises with virus nsPs in replication complexes. By using /jv
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113 deep sequencing, we observed an asymmetric distribution of SFV-derived viRNAs s
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114 with areas of high viRNA production (hot spots) and no/low frequency of viRNA r
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115 production (cold spots) along the length of the viral genome and antigenome, with a n

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116 slight bias towards production of genome-derived viRNAs. Infection of Ae. p
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117 albopictus- and Ae. aegypti-derived mosquito cells resulted in almost identical viRNA ,
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118 production patterns. Hot spot viRNAs, present at high frequency during infection, 9

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119 were found to have only poor antiviral activity compared to cold spot viRNAs. This
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120 points towards a nucleic acid-mediated RNAi decoy mechanism protecting against s
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121 antiviral RNAi. Bioinformatic analysis of RNA secondary structures and pairing in
122 the SFV genome (by MFE [minimal free energy]-based thermodynamic large scale
123 secondary structure prediction and StructureDist pairing/secondary structure
124 prediction) suggests no general correlation between frequency or location of viRNA
125 generation and putative RNA structures in the SFV genomic RNA. This strengthens
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126 the hypothesis that the origin and production of alphavirus viRNAs is linked to
127 dsRNA generation during replicative processes rather than secondary structures in the
128 viral genome.
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130 Materials and Methods
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132 Cells, viruses and infection. o
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133 Ae. albopictus-derived U4.4 and Ae. aegypti-derived Aag2 mosquito cells were grown lo
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134 at 28ºC in L-15 medium with 10% fetal calf serum and 8% tryptose phosphate broth. e
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135 Amplification and titration of SFV (strain SFV4) and clones derived from this virus o
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136 and infection of U4.4 cells were carried out as described previously (3). Briefly, SFV4
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137 or derived recombinant viruses were grown in BHK-21 cells in GMEM with 2% /jv
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138 newborn calf serum (37ºC, 5% CO atmosphere). Details of reporter viruses (Fig. 1) s
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139 used can be obtained from the authors. Viruses were purified from supernatant as r
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140 described previously (12). Virus (resuspended in TNE buffer) was titrated by plaque n

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141 assay on BHK-21 cells. For infection of mosquito cells, viruses were diluted in PBSA p
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142 (PBS with 0.75% bovine serum albumin); cells were infected at 28ºC for 1 h and ,
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143 washed twice to remove any unbound particles. 9

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144
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145 Transfection of hot spot/cold spot-derived viRNA mimic siRNAs. s
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146 siRNA mimics of SFV viRNAs were produced by Ambion (Table 1). siRNAs for
147 Renilla luciferase (RLuc), and negative control siRNA (#1) were obtained from
148 Ambion (AM4630, AM4611). Approximately 1.625x105 U4.4 or 1.6x105 Aag2
149 cells/well were grown in 24-well plates. Before transfection, medium was replaced by
150 fresh complete medium. siRNAs (final concentration 10 nM) were mixed with 1
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151 µl/well Lipofectamine 2000 (Invitrogen) in OPTIMEM according to the
152 manufacturer’s instructions. 100 µl of the nucleic acid/Lipofectamine 2000 complexes
153 were added to 400 µl of medium in each well and incubated for 5 h at 28ºC. After
154 transfection, cells were washed twice to remove liposomes, and complete medium
155 was added. Where indicated, SFV infection was carried out 24 h after siRNA
156 transfection.
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157 o
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158 Small RNA isolation and deep sequencing. lo
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159 Total RNA was isolated from U4.4 or Aag2 cells using Trizol (Invitrogen). 6.5x105 e
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160 U4.4 or 8.6x105 Aag2 cells grown in 6-well plates were infected with SFV (m.o.i. 10). ro
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161 Total RNA was extracted at 24 h post-infection; concentration and purity were
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162 determined using a NanoDrop 1000 (Thermo Scientific). All subsequent RNA /jv
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163 processing was performed at GenePool, University of Edinburgh s
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164 (http://genepool.bio.ed.ac.uk/). Small RNAs below 44 nt were purified from a 15% r
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165 TBE-Urea polyacrylamide gel, and RNA adapters ligated before sequencing according n

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166 to the manufacturer’s instructions (Illumina Inc., USA). Small RNAs were then p
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167 sequenced using a Illumina Solexa Genome Analyzer. Small RNAs derived from ,
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168 uninfected cells were sequenced as a control. To identify SFV-derived viRNAs, small 9

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169 RNA sequences were compared to the SFV4 reference genome/antigenome sequence
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170 (GenBank accession number: X04129) corrected by recent re-sequencing of SFV4 s
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171 (GenBank accession number: AJ251359) (compiled by A. Merits, personal
172 communication) using SOAP software (with the help of GenePool bioinformatics
173 support) (29). Small RNA data as used in figures within this paper are shown in
174 Supplementary Tables 1-3 (Excel tables) which contain viRNA sequences mapping to
175 the SFV genome or antigenome and both types of viRNAs are separately listed in
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176 labelled sheets within each Excel table; (+) indicates viRNAs mapping to SFV genome;
177 (-) indicates viRNAs mapping to SFV antigenome (sequences complementary to
178 antigenome viRNAs are shown in 5’ to 3’ orientation to simplify mapping; viRNA
179 sequence is complement, reading from left to right 3’ to 5’). viRNA location/starting
180 position on SFV genome or antigenome (column B), sequence (column C), frequency
181 (number of reads; positions from which no viRNA is generated are not shown and
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182 listed as 0 reads in Table 1) (column D) and length (nt) (column E) are indicated in o
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183 each sheet. Supplementary Table 1: SFV viRNAs from U4.4 cells used in Figs. 3, 6 and lo
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184 Supplementary Fig. 2. Supplementary Table 2: SFV viRNAs from U4.4 cells used in e
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185 Fig. 5, Supplementary Fig. 1, and Supplementary Fig. 2. Supplementary Table 3: SFV o
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186 viRNAs from Aag2 cells used in Figs. 4, 5 and Supplementary Fig. 1.
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187 /jv
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188 Luciferase assays. s
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189 Cells were lysed in Passive Lysis Buffer (Promega) and Renilla luciferase (RLuc) r
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190 activities measured using a Dual Luciferase assay (Promega) on a GloMax 20/20 n

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191 luminometer. p
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192 ,
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193 RNA secondary structure prediction. 9

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194 The existence and position of RNA secondary structures in the SFV genomic RNA
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195 were predicted by thermodynamic folding energy calculations that used comparative s
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196 sequence data from the available SFV complete genome sequences (SFV4 as above
197 and GenBank accession numbers EU350586, DQ189086, Z48163). This was
198 performed using the program UNAFold (62). For calculation of folding energies,
199 sequences in the SFV alignment were split into sets of 150 base fragments
200 incrementing by 1 base across the genome. Folding energies were compared with
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201 those of 50 copies of each fragment whose sequence order was scrambled using the
202 algorithm NDR that preserves dinucleotide frequencies of the native sequence
203 automated using the program Folding Energy Scan as implemented in the
204 Simmonic2005 v1.8 package (50). Minimum free energies (MFEs) of each native
205 sequence fragment were compared with the mean MFE of the NDR-scrambled
206 controls to produce a MFE difference (MFED) as previously described (51). MFED
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207 values for each fragment were averaged across the 4 available SFV complete genome o
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208 sequences. lo
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209 Prediction of specific structures using the available comparative sequence data for e
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210 SFV used the program StructureDist in the Simmonics package. This program o
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211 compares minimum energy structures from sequence datasets and computes
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212 phylogenetically conserved pairings and secondary structures. Results were expressed /jv
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213 in the form of pairing frequencies, which is calculated from the proportion of pairwise s
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214 comparison of connect files for each sequence with the same pairing predictions as r
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215 previously described (51). n

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216 p
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217 Immunostaining. ,
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218 U4.4 cells were fixed with 4% paraformaldehyde. After 2 washes (in PBS), cells were 9

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219 permeabilised with 0.3% Triton-X100 in PBS for 20 minutes and washed twice. After
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220 blocking with CAS block (Invitrogen), J2 antibody (Scicons, Hungary) (in CAS s
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221 block; 1:400) was added, followed by three washes. Incubation with secondary
222 antibody (sheep anti-mouse biotinylated IgG [Vector Labs] in CAS block) was
223 followed by three washes before addition of streptavidin-conjugated Alexa Fluor 594.
224 After a further two washes, slides were mounted with mounting medium (Vector
225 Laboratories) and images acquired (Zeiss AxioSkop confocal microscope).
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226 Results
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228 SFV infection-induced dsRNA co-localizes with virus replication complexes.
229 U4.4 mosquito cells have functional antiviral immune responses and resemble
230 infectivity of the mosquito; the acute phase of SFV infection in these cells lasts up to
231 24 h post-infection (p.i.) (3, 15, 35). dsRNA is produced in SINV-infected
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232 mammalian and mosquito cells (53). To determine the relationship between o
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233 alphavirus replication complexes and dsRNA (the trigger of immune responses) in lo
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234 mosquito cells, Ae. albopictus-derived U4.4 cells were infected with recombinant e
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235 SFV4 encoding a nsP3-eGFP fusion protein, SFV(3F)4-eGFP (55) (Fig. 1). The nsP3- o
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236 eGFP fusion marks (although not exclusively) viral replication complexes. At 10 and
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237 24 h p.i., cells were stained with anti-dsRNA antibody J2, which recognises dsRNA of /jv
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238 more than 40 bp in length (48, 60). nsP3-eGFP fusion protein and dsRNA co- s
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239 localized into punctate cytoplasmic complexes (Fig. 2) throughout the acute phase of r
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240 infection, a distribution consistent with virus replication on cytoplasmic vacuoles as n

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241 described for replication of SFV and other alphaviruses in vertebrate and invertebrate p
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242 cells (15, 16, 45, 52). This shows that the substrates for dsRNA pattern recognition ,
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243 are predominantly localised to virus replication vesicles. 9

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244
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245 The SFV-induced antiviral RNAi response: characterization and analysis of s
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246 viRNAs.
247 SFV-derived viRNAs generated during infection of Ae. albopictus-derived U4.4 cells
248 were characterized 24 h p.i. by deep sequencing. At a m.o.i. of 10, all cells in the
249 culture were infected with SFV synchronously; this is different from infection of live
250 mosquitoes where a mix of viRNAs from cells at different stages of infection is