Transgene-assisted genetic screen identifies rsd-6 and novel genes as key components of antiviral

52 Pages·2017·1.81 MB·English

JVI Accepted Manuscript Posted Online 27 June 2018
J. Virol. doi:10.1128/JVI.00416-18
Copyright © 2018 Long et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
1 Transgene-assisted genetic screen identifies rsd-6 and novel genes as key components
2 of antiviral RNAi in Caenorhabditis elegans
3 Tianyun Long*, Fei Meng* and Rui Lu#
4 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803,
5 USA D
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6 *These authors contributed equally to this work n
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7 #To whom correspondence should be addressed. E-mail: [email protected] d
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8 fr
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9 Abstract h
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10 RNA interference (RNAi) is a widespread antiviral mechanism triggered by :
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11 virus-produced double-stranded RNAs (dsRNAs). In Caenorhabditis elegans antiviral i.a
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12 RNAi involves a RIG-I-like RNA helicase, termed DRH-1 (dicer related RNA helicase 1), .
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13 that is not required for classical RNAi triggered by artificial dsRNA. Currently, whether /
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14 antiviral RNAi in C. elegans involves novel factors that are dispensable for classical D
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15 RNAi remains to be an open question. To address this question, we designed and carried e
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16 out a genetic screen that aims to identify novel genes involved in worm antiviral RNAi. e
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17 By introducing extra copies of known antiviral RNAi genes into the reporter worms we 2
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18 managed to reject alleles derived from 4 known antiviral RNAi genes, including DRH-1 8

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19 coding gene, during the screen. Our genetic screen identified altogether 25 alleles which g
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20 were assigned to 11 candidate genes and 2 known antiviral RNAi genes through genetic s
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21 complementation tests. Using mapping-by-sequencing strategy we identified one of the
22 candidate genes as rsd-6, a gene that helps maintain genome integrity through an
23 endogenous gene silencing pathway but was not known to be required for antiviral RNAi.
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24 More importantly we found that two of the candidate genes are required for antiviral
25 RNAi targeting Orsay virus, a natural viral pathogen of C. elegans, but dispensable for
26 classical RNAi. Since drh-1 is so far the only antiviral RNAi gene not required for
27 classical RNAi, we believe that our genetic screen led to identification of novel worm
28 genes that may target virus-specific features to function in RNAi. D
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29 n
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30 Importance e
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31 In nematode worms drh-1 detects virus produced double-stranded RNA (dsRNA) r
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32 thereby to specifically contribute to antiviral RNA silencing. To identify drh-1-like genes h
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33 with dedicated function in antiviral RNAi we recently carried out a genetic screen that :/
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34 was designed to automatically reject all alleles derived from 4 known antiviral silencing a
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35 genes, including drh-1. Of the 11 candidate genes identified we found two of them to be .o
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36 required for antiviral silencing targeting a natural viral pathogen of C. elegans, but not for o
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37 classical RNA silencing triggered by artificial dsRNA. We believe that these two genes D
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38 are novel components of worm antiviral RNAi, considering the fact that drh-1, the only m
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39 known antiviral RNAi gene that is dispensable for classical RNAi. This genetic screen r

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40 also identified rsd-6, a gene that maintains genome integrity under unfavorable condition, 2
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41 as key regulator of worm antiviral silencing, demonstrating an interplay between antiviral
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42 immunity and genome integrity maintenance. g
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43 t
44 Keywords: Classical RNAi, antiviral RNAi, siRNA, biased genetic screen
45
46
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47 Small interfering RNAs (siRNAs) processed from virus-derived double-stranded
48 RNA (dsRNA) mediate potent antiviral RNA interference (RNAi) in diverse organisms
49 (8). Mechanistic studies of antiviral RNAi have led to the identification of several key
50 factors involved in this process. Typically, an endoribonuclease III termed Dicer
51 processes viral dsRNA into siRNAs to initiate antiviral RNAi (31). Subsequently, an D
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52 Argonaute protein, a type of endoribonuclease with RNase H-like fold, recruits n
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53 virus-derived siRNA (vsiRNA) as sequence guides and cleaves viral transcripts d
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54 containing complementary sequence. dsRNA binding proteins also contribute to antiviral fr
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55 RNAi by facilitating viral dsRNA processing or vsiRNA loading into Argonaute proteins h
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56 (30, 33, 39). In plants and nematodes antiviral RNAi is further amplified through the :
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57 production of secondary vsiRNAs by RNA-dependent RNA polymerase (2, 11, 15, 41). i.a
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58 Recently, antiviral RNAi was observed in undifferentiated mammalian cells and appears .
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59 to provide protection against the attack of lethal viral pathogen for suckling mice (23, 27). /
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60 Since viral diseases are often the result of perturbation of host antiviral RNAi in diverse D
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61 organisms (6, 7), mechanistic study of antiviral RNAi holds the promise to develop novel e
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62 antiviral strategies. e
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63 Orsay virus naturally infects Caenorhabditis elegans nematodes (10), providing 2
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64 an ideal genetic model system for the study of virus-host interactions, including antiviral 8

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65 RNAi. Genetic and biochemical analyses suggest that antiviral RNAi in C. elegans is g
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66 initiated by the worm Dicer DCR-1, which processes viral dsRNA into primary vsiRNAs, s
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67 predominately 23 nucleotides in length (21, 25, 33). Efficient processing of viral dsRNA
68 by DCR-1 requires a dsRNA-binding protein termed RDE-4 (RNAi defective 4) (24, 33,
69 39, 43). The primary vsiRNAs are then loaded into RDE-1, one of the worm Argonaute
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70 (Ago) proteins that has slicer activity (24, 33, 43, 45). Instead of cleaving viral transcripts
71 with matching sequence, RDE-1 loaded with primary vsiRNA activates, probably with
72 help from co-factors, RRF-1, a worm RNA-dependent RNA polymerase (RdRP) (37, 45).
73 Subsequently, activated RRF-1 synthesizes secondary siRNAs in a DCR-1-independent
74 manner (1, 2, 15, 29, 36). Unlike primary vsiRNAs produced by DCR-1, secondary D
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75 vsiRNAs are single-stranded RNA molecules of 22 nucleotides long and carry a n
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76 tri-phosphate group at the 5’ end (12, 29, 36). d
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77 Antiviral RNAi in C. elegans also requires DRH-1 (Dicer-like RNA helicase 1) and fr
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78 DRH-3, two RIG-I-like RNA helicases (RLHs) that are not conserved in fungi, plants or h
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79 insects (25, 47). Previously we found that the C-terminal regulatory domain of human :
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80 RIG-I protein, which contributes to virus sensing in mammalian innate immunity (18, 20, i.a
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81 22, 26), can functionally replace the corresponding domain in DRH-1, suggesting a role .
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82 of DRH-1 in virus sensing (15). Consistently, virus-derived vsiRNAs were found to be /
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83 significantly reduced in drh-1 mutants (2, 15). Currently, it remains largely unknown D
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84 whether DRH-1-mediated virus sensing involves additional factors and how the function e
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85 of DRH-1 is regulated in response to virus invasion. Although sharing sequence e
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86 homology and domain structure with DRH-1, DRH-3 appears to function downstream of 2
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87 DRH-1 and is required for the production of secondary vsiRNA (2, 15). DRH-3 also 8

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88 plays an essential role in worm development (9). Currently exactly what DRH-3 does in g
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89 antiviral RNAi remains largely unknown. s
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90 RSD-2 is another key component of worm antiviral RNAi and appears to be
91 conserved only in the nematode kingdom (16, 25, 40). Previously we have shown that
92 RSD-2 contributes to both RDE-4-dependent and RDE-4-independent antiviral RNAi
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93 (16). Consistent with a prior study on classical RNAi (46), we found that vsiRNAs can by
94 readily detected by northern blot in asd-2 mutants with a size distribution similar to that
95 in rde-1 and rrf-1 mutants, suggesting that ASD-2 is dispensable for the biogenesis of
96 primary vsiRNAs and contributes to the amplification of antiviral RNAi by facilitating
97 the production of secondary vsiRNAs (16). Recently, a Vasa ATPase-related protein D
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98 termed RDE-12 was also found to contribute to antiviral RNAi probably by enabling the n
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99 production of secondary vsiRNA (34). d
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100 Previously we have shown that high-level viral replication in drh-1 mutants led to the fr
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101 production of vsiRNAs at low level (15). This observation together with a recent report h
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102 suggests a drh-1-independnet mechanism for the production of vsiRNAs in C. elegans (4). :
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103 Notably, these vsiRNAs are capable of mediating potent silencing of a homologous i.a
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104 transgene (15). Similarly, silencing of transgene but not homologous virus can also be .
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105 triggered in drh-1 mutants when RNAi is induced using artificial dsRNA (15). These /
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106 findings together suggest that viruses are more resistant to RNAi compared to D
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107 homologous cellular transcripts and as such additional genes may be required for antiviral e
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108 RNAi. Apparently, these antiviral-specific genes can only be identified in genetic screens e
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109 where replicating viruses are used as reporter of loss of RNAi. 2
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110 Orsay virus is a plus-strand RNA virus with a bipartite genome that resembles 8

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111 nodavirus in term of structure and sequence homology. Orsay virus only infects g
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112 intestine cells of C. elegans and such a tissue-specific infection pattern remains s
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113 unchanged when the virus was delivered into wild type worms or RNAi defective
114 mutants as a transgene known to be active in non-intestine cells (19). These observations
115 suggest that there is a receptor- and RNAi-independent mechanism that restricts systemic
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116 spreading of Orsay virus in C. elegans. Orsay virus replicates at low levels in wild type
117 N2 worms but accumulates to high levels in RNAi defective mutants or in the presence of
118 a functional RNAi suppressor (2, 14-16). These observations together with the fact Orsay
119 virus was originally isolated from a worm isolate naturally defective in antiviral RNAi
120 suggest that Orsay virus has very weak, if at all, activity in RNAi suppression (10). D
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121 Currently a modified Orsay virus derivative that would allow for the visualization of loss n
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122 of antiviral RNAi in C. elegans is still lacking. Thus, genetic screens that aim to identify d
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123 novel antiviral RNAi genes through genetic screen will need to seek alternative model fr
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124 virus as loss of antiviral RNAi reporter. h
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125 To identify novel genes with dedicated function in antiviral RNAi we have recently :
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126 carried out a large-scale genetic screen that utilized a flock house virus (FHV) replicon as i.a
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127 the loss of RNAi reporter (25). By integrating extra copies of four known antiviral RNAi .
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128 genes into the reporter transgene array we expected to automatically reject genetic alleles /
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129 derived from those four genes during genetic screen. Upon completing such a biased D
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130 genetic screen we isolated altogether 25 genetic alleles that were assigned into 11 e
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131 candidate genes and two known RNAi genes. Most importantly, we found that two of the e
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132 candidate genes are required for antiviral RNAi targeting Orsay virus but dispensable for 2
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133 classic RNAi. Since drh-1 alleles have been excluded during the screen we believe that 8

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134 these two candidate genes are novel requirement of worm antiviral RNAi. Using g
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135 mapping-by-sequencing strategy we also identified one candidate gene as rsd-6. rsd-6 s
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136 plays a role in endogenous gene silencing that help maintain genome integrity but was
137 not known to be required for antiviral RNAi. Our study thus revealed an interplay
138 between antiviral immunity and endogenous gene silencing pathway that maintains
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139 genome integrity.
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141 Results
142 The design of a biased genetic screen for the identification of C. elegans genes
143 required for antiviral RNAi D
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144 FHV is a member of the Nodaviridae family. Although not a pathogen of nematode lo
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145 worms, when delivered as a transgene, FHV replicates and triggers potent antiviral RNAi d

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146 in C. elegans (25), making it an alternative model virus for the study of RNAi mediated m

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147 virus-host interaction in C. elegans (13-16, 24, 42). Most importantly, an FHV RNA1 tp
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148 derivative, named FR1gfp, has been successfully developed as a reporter for loss of i.
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149 antiviral RNAi in C. elegans (Figure 1 A) (25). FR1gfp features an eGFP coding .
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150 sequence in place of B2 coding sequence and produces bright green fluorescence in o/
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151 worm mutants defective in RNAi. Thus, RF1gfp combined with large-scale genetic e
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152 screen will allow for the identification of novel genes involved in antiviral RNAi.
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153 So far altogether 10 C. elegans genes, including dcr-1, drh-1, drh-3, mut-7, rde-1, r

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154 rde-2, rde-4, rde-12, rrf-1 and rsd-2, have been implicated in antiviral RNAi through 2
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155 genetic analysis (2, 15, 16, 24, 25, 33, 34, 43). Since most of these genes, with exception
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156 of dcr-1, drh-3 and mut-7, are dispensable for worm development a conventional forward g
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157 genetic screen that aims to identify novel genes involved in antiviral RNAi may t
158 inevitably and repetitively pick up loss of function alleles derived from some of these
159 genes.
160 In C. elegans, gonad microinjection of target gene constructs often leads to the
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161 formation of large transgene arrays that contain many copies of target genes (28). Since
162 the formation of those large transgene arrays does not rely on the sequence homology
163 shared between the injected genes it is possible to generate a large transgene array that
164 contains many different transgenes. This observation suggests that we can co-deliver the
165 FR1gfp reporter and several transgenes corresponding to known antiviral RNAi genes D
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166 into the worm strain to be used for genetic screen. Since the chance to simultaneously n
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167 mutate both the transgenes and corresponding endogenous genes will be extremely low, d
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168 considering the fact that most of the transgenes will be delivered in multiple copies, fr
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169 genetic screens utilizing this novel worm strain will automatically reject null alleles h
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170 derived from the known antiviral RNAi genes chosen to be excluded during the screen. :
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171 Therefore, to better understand worm antiviral RNAi, we have developed an i.a
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172 experimental strategy, as shown in Figure 1, for identifying and characterizing worm .
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173 genes with dedicated function in antiviral RNAi. In this strategy, a reporter transgene /
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174 array is created to contain both a heat inducible FR1gfp transgene and transgenes D
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175 constitutively expressing four of the known antiviral RNAi genes, drh-1, rde-1, rde-4 and e
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176 rsd-2 (Figure 1 A). The FR1gfp transgene serves as loss of antiviral RNAi reporter e
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177 whereas the other 4 transgenes ensure that no loss of function alleles corresponding to 2
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178 these genes will be isolated during the genetic screens. Following the genetic screen the 8

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179 candidate mutants will be subjected to classical RNAi test to see whether they g
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180 specifically contribute to antiviral RNAi (Figure 1 B). s
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182 Generation of a reporter transgene array for biased genetic screen
183 To generate a worm strain that contains both a heat inducible FR1gfp transgene
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184 and transgenes corresponding to drh-1, rde-1, rde-4 and rsd-2, we combined six plasmid
185 constructs, as shown in Figure 1A, and injected them into rde-1;rde-4 double mutants
186 that already contain a heat inducible FR1gfp transgene. This microinjection led to the
187 production of 5 transgenic lines that contain transmissible extrachromosomal arrays. To
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188 find out whether the rde-1 and rde-4 transgenes in these extrachromosomal arrays are n
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189 functional we checked the expression of green fluorescence in the transgenic animals d
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190 after heat induction. Although few worms occasionally showed green fluorescence in fr
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191 pharyngeal tissue none of the transgenic animals, marked by the red fluorescence in h
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192 head, produced whole-body green fluorescence (Figure 2A), confirming that both rde-1 /jv
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193 and rde-4 transgenes in those extrachromosomal transgene arrays are functional. To sm
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194 generate a reporter strain for genetic screens we treated one of the transgenic lines with rg
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195 gamma ray irradiation and screened the F2 worms for integrated transgene arrays. This
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196 led to the identification of 3 integrated transgene arrays in total. By checking the e
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197 production of green fluorescence in response to heat induction we confirmed that all of e
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198 these integrated transgene arrays contain functional rde-1 and rde-4 transgenes. We chose 2
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199 one of the integrated transgene arrays, termed ty48, for further characterization mainly b
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200 because worms carrying this transgene array are free of any developmental defects. u
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201 After introducing the ty48 transgene array into non-transgenic rde-1;rde-4 double
202 mutants through outcross we performed northern blot analyses to detect the replication of
203 FR1gfp and Orsay virus in the resulting worm strain. As shown in Figure 2 B and C,
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204 neither of the viruses was able to replicate at high level and, accordingly, no whole-body
205 green fluorescence was observed after heat induction (data not shown). However, when
206 rde-1;rde-4 double mutants carrying the ty48 transgene array were fed with E. coli food
207 expressing either rde-1 or rde-4 dsRNA, enhanced Orsay virus replication was detected
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208 through northern blot analyses (Figure 2D), suggesting that the restoration of antiviral n
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209 RNAi in the double mutants is indeed due to the introduction of functional rde-1 and d
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210 rde-4 transgenes. As shown in Figure 2E, rde-4 dsRNA feeding led to the production of fr
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211 whole-body green fluorescence in the double mutants after heat induction, suggesting that h
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212 the FR1gfp transgene was successfully integrated into the ty48 transgene array. /jv
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213 sm
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214 Both drh-1 and rsd-2 transgenes are functional in the ty48 transgene array o
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215 To find out whether drh-1 and rsd-2 transgenes have also been successfully o
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216 integrated in the transgene array ty48, we transferred ty48 into drh-1 and rsd-2 mutants e
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217 through outcross and checked FR1gfp replication in the resulting worm strains after heat m
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218 induction. We found that, as shown in Figure 3A, the ty48 transgene array successfully r
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219 restored antiviral RNAi in drh-1 and rsd-2 mutants, leading to the suppression of FR1gfp 0
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220 replication. Moreover, no whole-body green fluorescence was observed in mutant worms b
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221 containing the ty48 transgene array (data not shown). We found that the replication of u
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222 Orsay virus was also suppressed to a level comparable to that in wild type N2 worms t
223 containing the same transgene array (Figure 3B). Apparently, the restoration of antiviral
224 RNAi in both mutants can only be ascribed to the introduction of the ty48 transgene array
225 since the replication of both FR1gfp and Orsay virus was restored when the expression of
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