Slicing of viral RNA guided by endogenous piRNAs triggers the production of responder and trailer piRNAs in Aedes mosquitoes

PIWI interacting (pi)RNAs are small RNAs mostly known to protect the genomes of animal germlines against transposable elements. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the release of a responder piRNAs via the ping-pong amplification cycle. Responder piRNA 3’ end formation by the endonuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of piRNAs that target transposons. Intriguingly, in Aedes aegypti mosquitoes, somatic piRNAs are produced from viral RNA, implying a role of viral (v)piRNAs in antiviral immunity. Knowledge on how vpiRNA 3’ ends are formed and whether viral RNA is subjected to trailer piRNA production, however, is lacking. To address these questions, we analyzed small RNA sequencing libraries from Ae. aegypti cells. We found that virus- and transposon-derived piRNAs have sharply defined 3’ ends, and that uridine residues are enriched directly downstream of dominant piRNA sequences, both of which are characteristic features of Zucchini-like cleavage of precursor piRNAs. Next, we designed a piRNA reporter system based on Sindbis virus recombinants that harbor target sites for abundant endogenous piRNAs. These piRNAs guide cleavage of the viral RNA, which is subsequently processed into abundant responder piRNAs. Using this reporter virus system, we identified the Ae. aegypti orthologs of Zucchini, which is required for sharp 3’ end formation of responder piRNAs, and Nibbler, a 3’-5’ exonuclease involved in the trimming of a subset of piRNAs and miRNAs. Furthermore, we found that cleavage of viral RNA triggers the production of trailer piRNAs, thus expanding the piRNA sequence pool that is able to target viral RNA. Our results have important implications for understanding how autonomous piRNA production from viral RNA can be triggered by just a few cleavage events by genome-encoded piRNAs.


INTRODUCTION
guided by a genomically encoded initiator piRNA triggers the production of additional trailer 105 piRNAs from the viral genome, thus increasing the pool of piRNAs targeting the newly 106 infecting virus. This mechanism may equip the Aedes piRNA pathway with an adaptive 107 immune response that is able to adapt to newly encountered and continuously mutating 108 viruses. sharp 3' ends and the nucleotide directly downstream of the 3' end tends to be a uridine (+1U 116 bias) (8,9). We examined whether these characteristics were present in our previously 117 generated small RNA deep sequencing libraries from Ae. aegypti Aag2 cells (24) infected 118 with Sindbis virus (SINV), a positive sense RNA virus of the Togaviridae family. We first 119 analyzed transposon-derived piRNAs and found that individual piRNAs, defined by a shared 120 5' end, generally had the same length ( Figure 1A). Specifically, for almost 60% of piRNAs the 121 dominant length made up more than 75% of sequenced reads. We selected those piRNAs 122 for which the dominant sequence length represented at least 75% of the reads and inspected 123 the identity of the nucleotides downstream of that most abundant piRNA isoform. We found 124 that the nucleotide position directly following the 3' end of the piRNA was biased for uridine 125 ( Figure 1B), strongly indicating that these piRNAs were generated by a mechanism that 126 resembles Zuc cleavage in Drosophila. Strikingly, sharp 3' ends and +1U biases were also 127 clearly visible for SINV-derived piRNAs, irrespective of the strand from which the piRNAs 128 were produced ( Figure 1C-D). These findings suggest that 3' ends of both (+) strand derived 129 vpiRNAs, which are predominantly Ago3-associated and (-) strand derived vpiRNAs, which 130 are mostly 34), are generated, at least in part, by Zuc-like cleavage events. 131 Interestingly, we also observe sharp 3' ends and +1U biases for piRNAs generated from  -derived piRNAs, defined by a shared 5' end. Shades of blue indicate the percentage of reads contributing to the indicated read length, white represents absence of reads in the specific size class. The number of unique piRNAs (nu) and the number of reads (nr) that underlie the heat map are indicated. A minimum of 20 reads/unique piRNA was required to be included in the analysis. (B) Nucleotide biases for the indicated nucleotide positions of transposon-derived piRNAs and the sequence at the genomic region directly downstream (+1 until +5) piRNA 3' ends. Only piRNAs from (A) that had a dominant length (at least 75% of reads) were considered in this analysis and only unique sequences were analyzed, disregarding read count for the individual piRNAs. (C-D) The same analysis as for A and B was applied to piRNAs mapping to SINV.

137
Genomically encoded piRNAs are able to trigger production of virus-derived 138 responder piRNAs 139 To study sequence determinants for vpiRNA 3' end formation, we designed a SINV-based 140 reporter system in which exogenous piRNA target sequences can be easily introduced. We 141 modified SINV such that a duplicated subgenomic promoter drives the expression of a non-142 coding RNA sequence that harbors a target site for an abundant Piwi5-associated initiator 143 piRNA ( Figure 2A, Figure S2A). Initiator piRNA-guided recognition of the target site should 144 trigger slicing by Piwi5 and subsequent processing of the resulting cleavage fragment into an 145 Ago3-associated responder piRNA through the ping-pong amplification cycle. We designed 146 viruses with target sites for two abundant endogenous Piwi5-associated initiator piRNAs: one 147 derived from the Ty3/gypsy LTR retroelement gypsy73 (g73 - Figure 2A), and a second 148 originating from an EVE of flaviviral origin (FV53, described in Supplemental text).

160
Responder piRNA size did not fully reflect the distance between the Piwi5 cleavage site and 161 the downstream uridine residue; while no clear differences in responder piRNA size are 162 observed between g73-25U and g73-28U viruses, increasing the 5' end-to-U distance to 30 163 nt (g73-30U) results in a more diffuse distribution of responder piRNA 3' ends ( Figure 2B).

164
These data suggest that downstream uridines may not be absolutely required for determining 165 the 3' ends of the reporter-derived responder piRNAs or that additional exonucleolytic 166 trimming of pre-piRNA 3' ends mask a putative endonucleolytic cleavage event directly 167 upstream of the uridine residues.

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To discriminate between these two possibilities, we generated small RNA deep sequencing 169 libraries from Aag2 cells infected with the various reporter viruses. We hypothesized that 170 even if a Zuc mediated cleavage event would be masked by exonucleolytic trimming, the 171 presence of downstream trailer piRNAs with 5' ends coinciding with the U residues would Figure 2. An endogenous piRNA is able to trigger production of virus-derived responder piRNAs (A) Schematic representation of recombinant Sindbis reporter viruses. The enlarged view depicts the reporter locus expressed under the control of a duplicated subgenomic promoter. This non-coding RNA harbors a target site for a Piwi5-associated initiator piRNA derived from the Ty3-gypsy73 retrotransposon (g73 -indicated in blue). Slicing of this target triggers the production of responder piRNAs (indicated in red) that are loaded into Ago3. The distance between the responder piRNA 5' end and the position of downstream uridine residues in the various viruses used in later experiments are shown. (B) Northern blot analyses of responder piRNA production in cells infected with the indicated reporter viruses. Numbers indicate the distance between the 5' end of the responder piRNA and the first downstream uridine residue. SINV 3' GFP is a virus without an initiator piRNA target site and serves as a negative control, as do mock-infected cells. The positions marking 24 and 33 nt are inferred from an EtBr-stained small RNA size marker. EtBr-stained rRNA serves as loading control. (C) Visualization of 5' ends of sense (red) and antisense (blue) piRNAs (24-33 nt) mapping to the reporter locus of the indicated viruses. Dashed lines indicate responder and initiator piRNA 5' ends and the positions of grey shading indicate the location of uridine residues on the sense strand of the various viruses. The red and blue numbers show piRNA counts (in reads per 10 6 miRNAs) that exceed the range of the y-axis; yellow arrowheads indicate the 5' ends of trailer piRNAs. (D) Size distribution of responder piRNAs produced from the various g73-viruses, as determined by small RNA seep sequencing. Read counts were normalized to the number of miRNAs in each library.
indicate Zuc-mediated endonucleolytic cleavage was responsible for generating the 173 responder piRNA 3' end. Mapping of piRNA 5' ends to the genomes of g73-targeted viruses 174 reveals that virtually no antisense piRNAs other than the initiator piRNAs map to the reporter 175 sequence ( Figure 2C). These initiator piRNAs trigger the production of highly abundant sense 176 responder piRNAs with characteristic 10 nt overlap of piRNA 5' ends, indicative of production 177 by ping-pong amplification ( Figure 2C). Responder piRNA size distribution for g73-targeted 178 viruses generally recapitulates the results from the northern blot analysis, with reduced 3' 179 end sharpness for the g73-30U virus ( Figure 2D). 180 We noted that responder piRNAs <26 nt in size are inefficiently produced from any virus 181 (including those with a responder 5' end-to-U distance of 25 nt: g73-25U and FV53-25/28U).

182
As responder piRNAs are expected to associate with Ago3, we hypothesize that Ago3 may 183 cover the first 25 nucleotides of pre-piRNAs, rendering them inaccessible to endo-or 184 exonucleolytic processing. Indeed, small RNA data from Ago3 immunoprecipitation indicate 185 a clear preference of Ago3 to bind piRNAs in the size range of 26-30 nt ( Figure S3A). In line 186 with this, responder piRNAs produced from all reporter viruses are predominantly 26-30 nt in 187 size ( Figure 2D).

188
Intriguingly, in viruses with a responder piRNA 5' end-U distance ≥28 nt, we uncover the 189 production of a putative trailer piRNA (indicated with yellow arrowheads in Figure 2C  Responder piRNAs are produced through ping-pong mediated slicing 200 We previously identified Ago3 and Piwi5 as the core components of the ping-pong 201 amplification loop in Ae. aegypti (24,34). We thus wanted to analyze which PIWI proteins are 202 responsible for the generation of the responder piRNAs from our reporter virus. As expected, 203 204 responder piRNA production was reduced upon knockdown of genes encoding the ping-pong 205 partners Ago3 and Piwi5 and, to a lesser extent Piwi6 ( Figure 3A, S4A). Surprisingly, 206 responder piRNA production was increased upon Piwi4 knockdown, which may be explained 207 by increased levels of the g73-derived initiator piRNA in Piwi4 knockdown ( Figure 3A). The  We next investigated targeting requirements for responder piRNA production. To this end, 211 we introduced target site mutations into the seed region (nt 2-8) and around the putative slice 212 site (nt 10-11, Figure 3C, S4C). Responder piRNA production was strongly depleted in 213 viruses in which mutations were introduced in the seed sequence (Mut 1-3, Mut 4-6 and Mut 214 7-9), compared to a virus bearing the intact target site (g73-28U, Figure 3D). Similarly, (D) Northern blot analysis of responder piRNA production in Aag2 cells infected with indicated (mutant) viruses. Responder piRNAs were detected using the 'minimal responder' probe that hybridizes to the last 18 nt at the 3' end of the responder piRNA (indicated in yellow in (C)), which are identical for all viruses. The dashed box denotes an area for which the contrast was adjusted to enhance weak responder piRNA signals (enhanced signal -middle panel). EtBr stained rRNA serves as loading control (bottom panel).
introducing mismatches around the slice site resulted in strongly reduced responder piRNA 216 production. As viral RNA levels are virtually unchanged between all viruses, reduced 217 responder piRNA production cannot result from differences in the amount of available 218 substrate ( Figure S4D). Weak responder piRNA production was observed in two seed 219 mutants (Mut 1-3 and Mut 4-6) as well as one slice site mutant (Mut10-11) ( Figure 3D). These 220 data suggests that low level piRNA slicing may occur even in the absence of full 221 complementarity in the seed region or the slice site, in line with findings in Drosophila (9) and 222 mouse (39).  Figure 4B).

235
To our surprise, we found that AAEL011385 contains a sizeable insertion directly 236 downstream of the catalytic HKD-motif ( Figure S5A). Similarly, Drosophila Zuc also contains 237 a (much smaller) insertion in the same location relative to mouse mitoPLD, suggesting it is a 238 variable region beyond Aedine mosquitoes. This non-conserved region is part of a helix that 239 sticks out of the Zuc core structure (36). Homology detection revealed that the large insertion 240 is conserved in the Culicidae family, but not in Drosophila, suggesting it is a mosquito-specific 241 insertion. Moreover, during cloning of the AAEL011385 gene, we found that the size of this 242 insertion is increased in Aag2 cells (32 additional amino acids; Figure S5A). 243 We next aimed to functionally validate AAEL011385 as the ortholog of DmZuc. Indeed,  (B) Confocal microscopy images of Aag2 cells expressing C-terminally 3xflag tagged Zuc. Mitochondria were stained using Mitoview green. On the right, enlargements of the areas indicated by dashed boxes in the image on the left are shown, with the nuclei oriented at the bottom. (C) Northern blot analysis of viral responder piRNA production in Aag2 cells upon dsRNA-mediated knockdown of Ae. aegypti PLDc_2 domain containing proteins and the PARN ortholog AAEL001426. Numbers indicate the VectorBase gene identifiers (without the AAEL0 prefix). The 24 and 33 nt size markers are inferred from an EtBr stained small RNA marker and rRNA stained. EtBr served as a loading control (same for (F)). (D) Viral responder piRNA (11585(+)) size distribution as determined in small RNA deep-sequencing libraries from dsLuc and dsZuc treated cells. Read counts were normalized to the number of miRNAs in each library. The inset shows the average responder piRNA read size in Luc-and Zuc knockdown libraries. Bars and whiskers represent mean and SD, respectively. (E) Read count of the responder piRNA in the reporter locus (left) and to the SINV genomic RNA (right), which is common for the three reporter viruses, in dsLuc and dsZuc treated Aag2 cells. Bars and whiskers show the mean +/-SD (left graph) and mean +/-SEM (right graph). (F) A sharpness score was attributed to all viral piRNAs upstream of the artificial reporter cassette (see materials and methods). The maximum score (log2(14) = 3.81) would be reached if 100% of reads that map to an individual piRNA had the same length. For each of the different reporter viruses the sharpness score for the top most 275 piRNAs were analyzed upon control and Zuc knockdown. The piRNAs were ranked according to the score in the control condition and the means within 11 bins of 25 piRNAs were determined (orange shade). The difference of piRNA score upon Zuc knockdown is plotted as mean ± SEM. A two-sided student's t-test was applied to each bin, to assess whether its mean was significantly different from zero. The null hypothesis was rejected at p<0.05. * P < 0.05 and ** P < 0.01 (G) Same as (F), but for transposon piRNAs.  Figure 4D). We propose that upon Zuc knockdown, the viral RNA 257 is cleaved downstream of the uridine residue, either by a hitherto unknown endonuclease or 258 by piRNA-PIWI ribonucleoprotein complexes, as in Drosophila (12). Subsequently, the 259 responder pre-piRNA may be trimmed, giving rise to mature piRNAs with more diffuse 3' ends 260 in a Zuc-independent manner.

261
To study the effects of Zuc knockdown on the 3' end sharpness of viral piRNAs outside of 262 the reporter locus in more detail, we assigned a sharpness score to every individual piRNA, 263 defined by a shared 5' end (see materials and methods for details). As expected, Zuc 264 knockdown significantly reduced sharpness scores, especially of those piRNAs that in control 265 conditions had the sharpest 3' ends and are therefore likely to be the most prominent Zuc 266 substrates ( Figure 4E). The same effect was observed for piRNAs that mapped to transposon 267 sequences ( Figure 4F). Moreover, Zuc knockdown resulted in an overall increase in size of 268 piRNAs produced from sense as well as antisense substrates of various origins, including  In Drosophila, piRNA 3' ends are generated by the concerted activities of two enzymes: the 288 endonuclease Zuc (7-9) and the 3' -5' exonuclease Nbr (12)(13)(14). Aside from its role in piRNA 289 3' end formation, Nbr is required for the trimming of microRNAs (miRNAs) (43,44). We set 290 out to identify the functional Ae. aegypti Nbr ortholog by predicting all DEDDy-type 3' -5' 291 exonuclease domain-containing proteins, which were used in a phylogenetic analysis along 292 with Drosophila Nbr (DmNbr). This analysis identified AAEL005527 as a one to one ortholog 293 of DmNbr ( Figure 5A). To verify that AAEL005527 is indeed the functional orthologue of 294 Drosophila Nbr, we assessed the effect of its depletion on trimming of two miRNA with 295 heterogeneous 3' ends in Ae. aegypti: miR-34-5p and miR-184 (45,46). It has previously been 296 shown that miR-34-5p undergoes extensive Nbr-mediated trimming in Drosophila (43,44).  To evaluate the role of exonucleolytic trimming on responder vpiRNA 3' end formation in Ae. 302 aegypti, we combined Nbr knockdown with infection using the g73-targeted reporter viruses.

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Knockdown was efficient and did not have major effects on viral RNA levels ( Figure S6A-B).

304
Interestingly, for all viruses tested, Nbr knockdown results in a specific loss of shorter (<27 305 nt) responder piRNA isoforms, but had no effect on larger isoforms ( Figure 5B, Figure S6C).

306
A similar reduction of shorter piRNA isoforms upon Nbr knockdown has previously been 307 observed in Drosophila (13,14). These findings suggest that mosquito Nbr may trim pre-308 piRNAs generated through a Zuc-mediated endonucleolytic cut, but that only a minor fraction 309 of such pre-piRNAs undergo trimming. Nbr-mediated trimming may be a general hallmark of 310 aging piRNAs, as has recently been proposed for miRNAs (47).

317
Targeting by an endogenous piRNA triggers trailer piRNA production. 318 While Ae. aegypti displays strong signatures of phased piRNA production (7), it is currently 319 unknown whether de novo produced RNA from cytoplasmic viruses is processed similarly through piRNA phasing. This is especially interesting as the genomes of Ae. aegypti and Ae. 321 albopictus mosquitoes contain large amounts of endogenous viral elements (EVEs). These 322 non-retroviral sequence elements are enriched in piRNA clusters and, accordingly, give rise 323 to abundant piRNAs (26), which may guide the slicing of cognate RNA from acutely infecting 324 viruses. It has been recently shown that piRNAs derived from EVEs are indeed able to target 325 and inhibit newly infecting viruses (31-33), yet, it remains unknown whether piRNA phasing 326 may serve as a mechanism to expand the vpiRNA pool after an initial cleavage by an 327 endogenous, possibly EVE-derived, piRNA.

328
Hence, we employed our viral reporter system to explore whether targeting by a genomically 329 encoded piRNA may trigger phased piRNA biogenesis from an acutely infecting virus. To this 330 end, we introduced an additional non-coding RNA sequence downstream of the g73-and 331 FV53-derived initiator piRNA target sites, which we termed the trailer cassette. To direct 332 sequential Zuc-mediated endonucleolytic cleavage, this cassette contains uridine residues at 333 regularly spaced intervals in an RNA sequence that is otherwise devoid of uridines (U interval 334 viruses, schematically shown in Figure 6C and Figure S7C). As a control, these uridines were 335 replaced by adenosine residues to create a trailer cassette completely devoid of uridines (U 336 desert viruses). In Aag2 cells infected with the g73 interval virus, we detected the first trailer 337 piRNA using northern blotting ( Figure 6A). Interestingly, we also observed the production of  Figure 6D, Figure S7D). Significantly fewer piRNAs were produced from the 351 trailer cassette in viruses containing a scrambled target site. In contrast, vpiRNA production Figure 6. Targeting of viral RNA by an endogenous piRNA triggers trailer piRNA production (previous page). (A) Northern blot analysis of the production of the first trailer piRNA from indicated viruses. As a control, the target site was scrambled to abolish targeting by the g73-derived piRNA. The remainder of the responder piRNA site as well as the trailer cassette are identical to the respective non-scrambled U interval and U desert viruses. As additional controls, a virus bearing an intact target site, but no trailer cassette (g73-28U), and a virus that contains no insert (SINV 3' EV) were used. The general structure of the g73 U interval virus is shown schematically in (C). rRNA stained with EtBr serves as a loading control. (B) RT-qPCR analyses of viral capsid RNA production in Aag2 cells infected with indicated viruses. All data are shown as a fold change relative to cells infected with a control virus lacking any insert (SINV 3' EV). Bars and whiskers show the mean and SD, respectively. (C) Schematic overview of the g73 U interval reporter virus. The inset shows a magnification of the non-coding reporter RNA expressed under control of the second subgenomic promoter. This reporter RNA contains a target site for a Piwi5-bound g73-derived initiator piRNA, which guides the production of an Ago3-associated responder piRNA. The downstream sequence makes up the trailer cassette and either contains regularly spaced uridine residues (g73U interval) or is completely devoid of uridine residues (g73U desert). Indicated in green is the first trailer piRNA, which was detected in (A).   Target sites for initiator piRNAs and reporter responder piRNA were introduced into the 391 recombinant Sindbis virus backbone to be expressed from a second subgenomic promoter.

392
The previously described SINV-GFP (50,51) was digested using XbaI to remove the GFP-  To introduce U-interval and U-desert reporter sequences, the g73-28U and FV53-25/28U 400 viruses were digested using NotI followed by ligation of annealed oligo's (see below).

405
Target site mutations were introduced into the g73-28U virus backbone by mutagenesis PCR, 406 using the primers shown below. PCR-products were DpnI-treated and In-fusion (Takara 407 Biotech) was using to circularize the plasmid for transformation. After verification of the 408 sequence by Sanger sequencing, viruses were grown as described previously (25).

530
The entire analysis was repeated for the six datasets of the g73-28 and g73-30 viruses and 531 the data was combined by calculating the mean + SEM of the ΔSsharp per bin obtained for 532 each type of reporter virus. To determine statistical significance, the ΔSsharp was tested 533 against the null hypothesis that there is no effect of Zuc knockdown on 3' end sharpness 534 scores (ΔSsharp = 0). A two-sided student's t-test was performed per bin and the hypothesis 535 was rejected at p<0.05. The sharpness score analysis for transposon mapping piRNAs was 536 performed with the 4400 most expressed piRNAs (400 piRNAs per bin).

RT-qPCR 539
For RT-qPCR analyses, DNaseI (Ambion)-treated RNA was reverse transcribed using    An EVE-derived piRNA has the potential to trigger responder piRNA production.

612
Apart from the g73 piRNA triggered reporter virus described in the main text, we generated 613 a second set of viruses bearing target sites for an abundant Piwi5-associated piRNA derived 614 from an EVE of flaviviral origin (FV53 (26), Figure S2A). As the target site for the FV53-615 derived initiator piRNAs can be recognized by two piRNA isoforms that align at their 3' end 616 and differ 3 nt in size, target cleavage may result in the production of two Ago3-bound 617 responder piRNAs isoforms that have an offset of 3 nt (Inset in Figure S2A).

622
For all viruses tested here, we observe abundant responder piRNAs as well as the production 623 of a first trailer piRNA (indicated with yellow arrowheads in Figure S2C). As the trailer piRNA instruct production of these additional sense piRNAs. 631 Figure S2. An EVE-derived piRNA triggers responder piRNA production from an acutely infecting virus RNA (A) Schematic depiction of the SINV-based viral reporter system in which responder piRNA production is triggered by an initiator piRNA derived from an endogenous viral element (FV53). As the Piwi5-associated FV53 initiator piRNA is expressed as two isoforms that align at their 3' end (blue and green lines), endonucleolytic cleavage may generate two Ago3-bound responder piRNAs (red lines) that differ 3 nt in size. The inset shows a part of the viral target RNA (red), the 5' ends of the two FV53-piRNA isoforms (27-mer: green and 30mer: blue) and the scissors represent their respective slice sites (green and blue).

Figure S4
635 Figure S4. PIWI-knockdown does not affect virus replication (A) RT-qPCR analyses of subgenomic capsid RNA abundance in the samples used in Figure 3A. RNA levels are shown as a fold change relative to dsLuc treated cells infected with g73-25U. Bars and whiskers show the mean and standard deviation (SD), respectively (same for B and D).
(B) RT-qPCR analyses of capsid RNA abundance in the indicated U4.4 (yellow) and Aag2 (blue) samples used in Figure 3B. Values shown are fold changes relative to WT cells infected with the SINV 3' EV control virus.
(C) Schematic representation of the g73-28U virus that was used to study the effect of target site mutations on responder piRNA production. Yellow shading indicates the area that is shown in Figure 3C.
(D) RT-qPCR analyses of capsid RNA levels in samples used in Figure 3D. All data are normalized to cells infected with the g73-28U scrambled virus.  Figure 4B, showing the responder piRNA size shift upon Zuc knockdown, analyzed for the three g73-derived piRNA targeted viruses separately. The X-axis represents the position on the northern blot from top to bottom, the Y-axis shows responder piRNA signal intensity. (C-D) RT-qPCR analyses of the knockdown efficiency of indicated genes (C) and viral capsid RNA levels (D). Gene expression levels are shown as fold change relative to dsLuc treated cells infected with the g73-25U virus. Bars and whiskers depict mean and SD, respectively. (E) Western blot analyses of Piwi4 and Piwi6 in the same Zuc-3xflag-IP material that was used in Figure 4H. (F) Average size of piRNAs (24-33 nt) derived from the indicated substrates in small RNA deep sequencing libraries of dsLuc and dsZuc treated Aag2 cells. The length profile of two piRNAs (tapiR1/2) involved in degradation of maternal transcripts during embryogenesis (58), is not affected by Zuc knockdown, suggesting that they are generated through an Zuc-independent, non-canonical mechanism. As virtually no antisense reads map to the tapiR1/2 locus, these data are not shown. Bars and whiskers represent mean and SD, respectively.

Figure S6. Nbr-and Zuc knockdown affect piRNA biogenesis (A-B)
RT-qPCR analyses of the knockdown efficiencies of the indicated genes (A) and viral capsid RNA levels (B). Expression values represent the fold change relative to dsLuc treated cells infected with the g73-25U virus. Bars and whiskers represent the mean +/standard deviation.
(C) Quantification of the northern blot signal in Figure 5B, showing the effect of Nbr knockdown on responder piRNA size during infection with the indicated viruses. The Y-axis represents responder piRNA signal intensity, the X-axis represents the position on the northern blot from top to bottom. Figure S7. An EVE-derived piRNA triggers trailer piRNA production downstream of its target site (previous page) (A) Northern blot analyses showing the production of the first putative trailer piRNA (marked in green in (C)) from the indicated viruses). As a control, the target site was scrambled to abolish targeting by the FV53-derived initiator piRNA. The remainder of the responder piRNA site, as well as the trailer cassette was identical to the U interval and U desert viruses. Additional controls include a virus that lacks the trailer cassette, but contains an FV53 piRNA-target site (FV25-28) and a control virus without any insert (SINV 3' EV). A schematic overview of the FV53 interval virus is shown in (C). rRNA stained by EtBr serves as loading control. (B) Capsid RNA levels in Aag2 cells infected with indicated viruses as determined by RT-qPCR. All capsid RNA levels are shown as a fold change relative to the virus lacking an initiator piRNA target site (SINV 3' EV). Bars and whiskers denote the mean and SD, respectively.
(C) Schematic overview of the FV53 U interval virus. In this virus, a duplicated subgenomic promoter drives the expression of a noncoding reporter RNA (shown in the magnification), which contains a target site for two isoforms of a Piwi5-bound FV53-derived initiator piRNA (shown in blue). Slicing of the reporter RNA in the ping-pong amplification loop thus may give rise to two differently sized Ago3associated responder piRNAs (shown in red). Downstream of this responder piRNA, the trailer cassette is located which either contains uridine residues at regularly spaced intervals (FV53 U interval) or is completely devoid of uridines (FV53 U desert). The first trailer piRNA which was detected in (A) is shown in green.
(D) Visualization of normalized counts of 5' ends of sense (red) and antisense (blue) piRNA-sized reads (24-33 nt) mapping to the noncoding reporter RNA. The 5' ends of the two FV53-derived initiator piRNA isoforms, as well as the 5' ends of putative responder piRNAs are indicated by dashed lines. Numbers indicate normalized read counts where they exceed the range of the y-axis and grey shading indicates the position of uridine residues.
(E) Quantification of the number of sense (red) and antisense (blue) piRNAs (in reads per 10 6 miRNAs) mapping to the trailer cassette (left) and genomic RNA (right) of the indicated recombinant Sindbis viruses. The areas of the viruses denoted as trailer cassette and genomic RNA are indicated in (C) in yellow and blue, respectively.

Figure S8
642 Figure S8. A model of piRNA-based adaptive immunity in mosquitoes Genomically encoded endogenous viral elements (EVE) give rise to a pool of Piwi5-associated initiator piRNAs that have the potential to target newly infecting viruses. Upon acute infection with a virus containing a cognate sequence, EVE-derived piRNAs trigger the production of Ago3-bound responder piRNAs from the viral RNA, which are generated and maturated by the combined activities of Zuc and Nbr. Additionally, targeting of the viral RNA by the ping-pong machinery prompts the processing of the downstream RNA into additional trailer piRNAs, thus expanding the piRNA sequence pool that is able to target viral RNA.