Dicing of intermediates during silencing of latent Drosophila

Alex Flynt, Na Liu, Raquel Martin, and Eric C. Lai1

Department of Developmental Biology, Sloan-Kettering Institute, 1017 Rockefeller Research Laboratories, 1275 York Ave, Box 252, New York, NY 10065

Communicated by Mark Ptashne, Memorial Sloan Kettering Center, New York, NY, January 7, 2009 (received for review November 26, 2008) Previous studies revealed roles for RNA interference (RNAi) in the NAs were preferentially generated from particular regions of the immediate cellular response to viral in plants, nematodes bipartite FHV genome. Unexpectedly, the abundant siRNAs de- and flies. However, little is known about how RNAi combats rived from latent were not effective in silencing reporters viruses during persistent or latent . Our analysis of small bearing complementary sequences. Moreover, although viral siR- RNAs cloned from Drosophila cells latently infected with Flock NAs were preferentially associated with the siRNA effector AGO2 House Virus (FHV) failed to reveal signatures of bulk degradation relative to the miRNA effector AGO1, bulk FHV-derived siRNAs of the viral genome. Instead, this ؉ strand virus specifically were unmethylated and did not substantially associate with either generated Dicer-2-dependent, 21-nucleotide siRNAs that derived in Argonaute. These data suggest that direct dicing of the replication equal proportion from ؉ and ؊ strands. Curiously, luciferase intermediate plays an important role during latent infection, and reporters that are fully complementary to abundant viral siRNAs hint at different activities of the RNAi pathway during acute were poorly repressed. Moreover, although the viral siRNAs that infection versus persistent infection. were incorporated into an effector complex associated with Ar- gonaute2, bulk FHV siRNAs in latently infected cells were not Results loaded into any Argonaute protein. Together, these data suggest Persistent Infection of Multiple Drosophila S2 Lines by Flock House that direct dicing of viral replication intermediates plays an impor- Virus. Recent examination of largescale small RNA sequence data tant role in maintaining the latent viral state. In addition, the denial indicated the persistent infection of an S2 stock (‘‘S2-NP,’’ from of bulk viral siRNAs from effector complexes suggests that criteria the laboratory of Norbert Perrimon) by Flock House Virus (FHV), GENETICS beyond the structural competency of RNA duplexes influence the a bipartite positive strand ϩ RNA virus (17). FHV RNA1 encodes assembly of functional silencing complexes. protein A, the viral RNA dependent RNA polymerase. It also generates RNA3 encoding B2, a dsRNA binding protein that was Argonaute2 ͉ Dicer-2 ͉ RNA interference ͉ virus proposed to mask the viral genome from the RNAi defense (2, 19, 20). The second segment of the FHV genome, RNA2, encodes the enetic analyses showed that core components of the RNA proteins. Ginterference (RNAi) pathway generate viral small interfering Although productive FHV infection induces and is RNAs (siRNAs) and restrict virus accumulation in flies (1–5), lethal in adult Drosophila and S2 cells (1, 2, 19), this virus persists worms (6–8), and plants (9, 10). Such studies indicate that the in S2-NP seemingly without affecting cell proliferation or survival. cellular defense to viral infection begins when double-stranded We performed Northern blot analysis to confirm the presence of RNA (dsRNA) viral genomes or replication intermediates are FHV genomic RNAs in these cells, and observed considerable cleaved by Dicer-class RNase III into small interfering accumulation of RNA2 and RNA3, but little of full length RNA1 RNAs (siRNAs). The viral siRNAs are incorporated into Argo- (Fig. S1). We collected several other S2 and S2Rϩ cells from various naute complexes that subsequently cleave and degrade viral coding laboratories and surveyed them for FHV. S2 cells from Ram RNAs, preventing completion of the viral lifecycle. To counteract Dasgupta’s laboratory (‘‘S2’’) and Phillip Zamore’s laboratory the RNAi defense, many viruses have evolved proteins that inhibit (‘‘S2-Z’’) were free of FHV (Fig. 1A and Fig. S1), but S2 cells from various components of the RNAi pathway, thus permitting their Gerald Rubin (‘‘S2-GMR’’) and S2Rϩ cells from Dasgupta’s lab- successful replication and/or infection (11). oratory (‘‘S2Rϩ’’) both harbored FHV. Therefore, persistent FHV In Drosophila, the RNAi and microRNA (miRNA) pathways are infections are fairly common among Drosophila laboratories. genetically distinct, but have substantial cross-talk (12). The canon- ical RNAi pathway uses Dicer-2 to process dsRNA into siRNAs, Canonical RNAi Factors Restrict Persistent FHV Infection. In principle, which are mostly loaded into Argonaute2 (AGO2) complex. deleterious mutations in persistent FHV strains might account for Ј AGO2-resident RNAs are then methylated at their 3 ends by the their reduced potency. For example, latent FHV might simply be Hen1 methyltransferase (13, 14). The miRNA pathway uses Dcr-1 mutated for the B2 suppressor, whose loss prevents FHV from to process premiRNA hairpins into mature miRNAs, which are mounting a productive infection of WT cells (1, 2, 19). However, mostly loaded into Argonaute1 (AGO1) complex; they remain assembly of the B2 ORF from the cloned small RNAs in 2 different unmethylated. Recent studies demonstrated that the sorting of strains (S2-GMR and S2Rϩ) revealed that it was intact, save for a diced small RNAs is influenced by the structural features of their few nucleotide changes with respect to the reference virus se- duplex precursors. Small RNAs from perfectly double stranded quence. Furthermore, ectopic expression of functional B2 into duplexes are favored to enter AGO2, whereas central bulges favor latently infected cells did not restore the replication of FHV. entry into AGO1 (15, 16). However, these rules do not entirely Exogenous B2 was able to bind to the viral genome (Fig. 1B), explain the types of RNAs that are resident in AGO1 and AGO2 suggesting that the latent strain retained the cis-acting sequences for (17, 18); therefore, there are presumably additional determinants that affect small RNA loading. Because the studies to date focused on the role of RNAi in Author contributions: A.F. designed research; A.F., N.L., and R.M. performed research; A.F., combating acute infection, little is known about its role during latent N.L., and E.C.L. analyzed data; and A.F. and E.C.L. wrote the paper. infection. To address this, we exploited cell lines that are persis- The authors declare no conflict of interest. tently infected by Flock House Virus (FHV). RNAi has a significant 1To whom correspondence should be addressed. E-mail: [email protected]. role in maintaining FHV latency, and the small RNA signatures of This article contains supporting information online at www.pnas.org/cgi/content/full/ this defense provided mechanistic insight. Dicer-2-dependent siR- 0813412106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813412106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 GFP dcr-1 dcr-2 ago1 ago2 dsRNA A B Gal4 His-Myc-B2 C

yc Input Input T7 RNA1 S2R+S2-Z S2-Z 1dpiS2-Z 3dpiS2-Z 6dpi His His M Myc * RNA2 T7 RNA1 RNA3

* FHV RNA2 (+) RNA2 U6 RNA3 1.0 0.46 3.10 1.30 2.99 RNA1

FHV RNA2 (-) 1.0 0.74 2.56 1.86 2.48 RNA2 1.0 0.66 1.70 1.35 1.90 RNA3

Fig. 1. Latent FHV infection is maintained by the RNAi pathway. (A) FHV transcripts were detected in S2Rϩ cells from the DasGupta laboratory, but not in a culture of S2 cells obtained from the Zamore laboratory (S2-Z). Addition of S2Rϩ conditioned media to S2-Z cells resulted in rapid and high level accumulation of FHV transcripts. (*), background hybridization to ribosomal RNA. (B) Coimmunoprecipitation of both ϩ and Ϫ strands of FHV RNA2 with His/Myc-tagged B2. Specificity of the interaction was assessed by background levels of FHV RNA2 recovered in the absence of transfected B2, or after immunoprecipitation with control T7 antibody. (C) Expression of FHV genomic RNAs after depletion of RNAi/miRNA pathway components. (Top) FHV transcripts were up-regulated after depletion of dcr-2 or ago2.(Middle) The blot was stripped and probed for U6 as a loading control; note the slightly unequal loading in the ago2 lane. (*), ribosomal RNA background. Bottom is a quantification of the viral transcripts, normalized first to the U6 level and then expressed as a ratio of the level in cells treated with GFP dsRNA.

B2 recognition. Thus, alterations in B2 do not appear to account for dicing into siRNAs. Because FHV positive strands outnumber the latency of FHV. negative strands by at least 50:1 (21), these cloning data suggest that We next used a functional test to report directly on the patho- FHV siRNAs were generated from a double-stranded replication genicity of latent FHV. We prepared conditioned media from intermediate. Notably absent were degradation fragments of the ϩ latently infected S2Rϩ cells and added it to cultures of naive S2-Z strand, which might have been expected to be present as a conse- cells. After as little as 24 h, newly infected cells showed signs of quence of host defense of the persistent FHV infection. In Dro- apoptosis, such as membrane blebbing. Northern blot analysis sophila cells, degradation fragments are rapidly phosphorylated revealed a dramatic accumulation of each of the FHV RNAs in the resulting in their cloning via 5Ј phosphate-dependent protocols newly infected cells (Fig. 1A). The abundance of FHV RNA2 and (22). Abundant cellular transcripts therefore generate short RNAs RNA3 was considerably higher in the newly infected cells than in of heterogeneous length that span the size window used for cloning latently infected cells, and importantly strong induction of RNA1 (23). In contrast, the vast majority of FHV reads were precisely was now seen. Thus, the latent FHV strain is capable of mounting 21 nt (Fig. 2A). robust infection and replication. The level of RNA1 in the newly Although the entire FHV genome is expected to be double- infected cells decreased substantially by 6 days postinfection, evi- stranded during ϩ strand replication, the majority of FHV siRNAs dence for a host defense to the viral infection. mapped to discrete regions of the virus. Most of the viral siRNAs Previous studies showed that components of the canonical RNAi were generated from near the 5Ј ends of both FHV genomic RNAs, pathway mediate defense against various single-stranded RNA and with certain internal regions also producing a substantial number of double-stranded RNA viruses (1–4). We tested whether RNAi siRNAs (Fig. 2 B and C). The spatial patterns of FHV siRNA activity was responsible for maintaining viral latency. We depleted mappings were highly reproducible between independent samples the core components of the RNAi and miRNA pathways (dcr-1, of S2Rϩ and of S2-GMR cells, and these different cell lines dcr-2, ago1, and ago2) in the latently infected S2Rϩ cells and assayed exhibited similar overall distribution of siRNAs along the FHV virus accumulation by Northern blot analysis. We observed signif- genome (Fig. S2). A recent analysis of small RNAs cloned from S2 icant accumulation of viral transcripts upon loss of Dicer-2 or cells acutely infected with FHV lacking the B2 product revealed a AGO2 (Fig. 1C). These data extend a recent quantitative rt-PCR similar spatial pattern of FHV-derived siRNAs (5). analysis of FHV RNA2 levels after knockdown of small RNA The coincidence of ϩ and Ϫ strand siRNAs provided additional pathway components (17). In particular, the increased abundance evidence of their origin from dicing of the replication intermediate, of full length RNA1 upon depletion of Dicer-2 or AGO2, as rather than from intramolecularly base-paired segments. We hy- determined by these Northern blot analysis experiments, reported pothesize that these siRNA hotspots represent sensitive points in directly upon the derepression of viral replication. the FHV genome to Dicer-2. Interestingly, we determined that dsRNA binding B2 suppressor protein bound both ϩ and Ϫ strands Characteristic Size and Spatial Distribution of Small RNAs Derived of FHV (Fig. 1C). Based on the viral siRNA cloning data, it seems from Latent FHV. We sought further insight into the molecular likely that B2 is bound to the replication intermediate. strategies by which small RNA pathways recognize and degrade these viral genomes. To do so, we analyzed small RNA sequences Latent FHV-Derived siRNAs Mediate Poor Repression of Complemen- from latently infected S2-GMR and S2Rϩ cells using the Illumina tary Targets. Based on the known requirement of AGO2 for viral platform. To ensure that the quantitative results from these se- defense (2, 3, 17, 19), a reasonable expectation is that viral siRNAs quencing experiments were reproducible, we analyzed 2 indepen- mediate the silencing of complementary viral transcripts in trans. dent small RNA libraries from each celltype yielding Ϸ30–60,000 To test this, we designed luciferase sensors containing 70 nt sense FHV reads per library. Because these libraries yielded 3–4 million and/or antisense segments of several regions of FHV RNA2 (Fig. matches to the reference genome, they contain Ϸ1–2% FHV reads 3A). These correspond to 170–240 bp (R1), and 530–600 bp (R3) (Table S1). from which large numbers of siRNAs are derived. We also cloned Size distribution analysis revealed FHV was predominantly pro- a companion region from 430 to 500 bp (R2) that did not yield cessed into 21 nt species, with similar overall numbers of sense and substantial numbers of siRNAs (Fig. 3B). Finally, we generated a antisense FHV reads (Fig. 2A). Their strong 21 nt bias suggested matched set of mutant sensors in which the viral sequence was that the majority of FHV-derived small RNAs are generated by mutated at every 4th position, so as to disrupt the possibility for

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2000 FHV RNA1 (+) Fig. 2. Features of FHV-derived small RNAs in latently infected S2Rϩ cells. (A) Predominantly 21 nt RNAs were ϩ Ϫ genomic position (nt) generated from both and strands of FHV, indica-

tive of siRNA processing. (B) Small RNAs mapped to GENETICS 0 FHV-RNA1. (C) Small RNAs mapped to FHV-RNA2. Dis- reads 1000 2000 3000 crete regions of the FHV genome generate abundant siRNAs. These graphs report the combined reads from FHV RNA1 (-) 2 independently-generated libraries; Fig. S2 shows that similar patterns were seen in the 2 datasets. More- 2000 over, the spatial distribution of siRNAs from the viral genome was similar between independent types of cultured cells bearing latent FHV infection.

either siRNA- or miRNA-like regulation by viral siRNAs. These and biochemical properties. FHV siRNAs derived from the 170– mutant sensors were used to normalize the expression of compan- 240 bp (R1) region of RNA2 in latently infected cells were detected ion wild-type reporters in FHV-infected (S2Rϩ and S2-NP) and on Northern blots using a combination of 4 LNA probes. We used FHV-free (S2) cells. this assay to examine viral siRNA accumulation after the depletion Active small RNA-mediated repression would yield ratios of of core miRNA/siRNA factors. The efficacy of knockdowns was wild-type to mutant sensor activities that are Ͻ1, but we would assessed by the behavior of a previously tested miRNA (bantam) expect their values to be equalized in cells lacking the cognate small and endo-siRNA (hp-CG4068B) (17, 18, 24, 25). As expected, RNAs. Surprisingly, the ratios of WT sensors complementary to the mature bantam levels were decreased after knockdown of dcr-1 or abundant siRNAs derived from R1 and R3 were nearly equivalent ago1, whereas the levels of mature hp-CG4068B were decreased to their mutant counterparts in latently infected cells (Fig. 3B). If after knockdown of dcr-2 or ago2 (Fig. 4A). anything, the sensor complementary to the extremely abundant R1 Using these validated knockdown samples, we observed that siRNAs was slightly elevated compared with its mutant control mature FHV-siRNAs were highly dependent on dcr-2, even moreso sensor. In addition, the normalized sensor values from the non- than hp-CG4068B. In contrast, FHV-siRNAs exhibited far less siRNA producing region 2 were not markedly different from the dependence on ago2 than did hp-CG4068B. This correlated with siRNA-complementary sensors, even though the level of R1 siR- the failure of FHV-derived siRNAs to mediate substantial target NAs was 370-fold that of R2 siRNAs. Finally, no substantial repression (Fig. 3), and suggested that the majority of FHV-derived differences were observed with R1 and R3 sensors in infected versus virus-free cells (Fig. 3B). siRNAs might not be loaded into functional effector complexes. To confirm the viability of these sensor assays, we performed There was an apparent increase in FHV-derived siRNAs after ago1 parallel experiments with WT and mutant sensors for the endo- knockdown. It is not clear whether this reflects an indirect effect of siRNA hp-CG4068B (24). As expected from previous studies (24), increased viral replication in the absence of this miRNA effector, we observed 3- to 5-fold repression of this sensor by endogenous or whether loading into AGO2 is improved if there is less AGO1 hp-CG4068B. The level of endogenous hp-CG4068B is similar to, present. or indeed much lower than, the various FHV siRNAs assayed, and To test for the presence of unloaded FHV siRNAs, we examined these quantitative comparisons were stable over multiple libraries the association of FHV-derived siRNAs with AGO1 and AGO2. A and cell types (Fig. 3 and Table S1). Therefore, these results indicate recent report of largescale sequencing of AGO1- and AGO2- that the abundant viral siRNAs produced during latent viral associated RNAs in S2-NP revealed that the incorporation of infection are largely ineffective at silencing complementary FHV-derived RNAs into AGO2 was strongly preferred to AGO1 transcripts. (17). The AGO2-IP dataset GSM280087 harbors 42,364 FHV- derived reads relative to 916,834 reads that mapped to the dm3 Bulk FHV-Derived siRNAs from Latent Infection Are Not Loaded into assembly of the Drosophila melanogaster genome (Ϸ4.41% si- Argonaute Proteins. We sought to explain the disconnect between FHV), whereas the AGO1-IP dataset GSM280088 contains only viral siRNA production and activity by examining their biogenesis 3567 FHV-derived reads compared with 2,094,408 reads that

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Fig. 3. FHV-derived siRNAs do not mediate effective target repression. (A) Three Ϸ70 nt regions of FHV-RNA2 (see Fig. 2C) were selected to generate WT renilla luciferase sensors. Companion mutant sensors contained mutations at every 4th position; sensors were made for both ϩ and Ϫ strands of region 3. The table shows the number of reads obtained from the 3 regions of FHV RNA2; the reads matching the endo-siRNA hp-CG4068B are shown for comparison. hp-CG4068B is slightly more abundant than R3ϩ siRNAs, similar in abundance to R3Ϫ siRNAs, and far less abundant than R1ϩ siRNAs. (B) FHV siRNA sensors were assayed in FHV infected and FHV-free cells. The ratios of normalized WT to mutant sensor activities were plotted, and standard error is depicted. In contrast to the hp-CG4068B sensor, which exhibited 3- to 5-fold repression compared with its mutant sensor (24), FHV siRNAs did not mediate substantial target repression. The dotted line provides a reference for the expected level of repression mediated by siRNAs whose expression is in the range of hp-CG4068B.

mapped to the Drosophila genome (Ϸ0.170% si-FHV). Thus, there whereas the endo-siRNA hpCG4068B was mostly associated with are Ϸ26 times more FHV RNAs in AGO2 than in AGO1. AGO2 (24, 25). To our surprise, although a minor fraction of To directly verify the preferred sorting of viral siRNAs to AGO2, FHV-derived siRNA was complexed with AGO2, consistent with we immunoprecipitated AGO1 or FLAG-tagged AGO2 from previous observations, bulk viral siRNAs from the ϩ or Ϫ strands S2Rϩ cells and probed their contents (Fig. 4B). As shown previ- did not associate with either Argonaute (Fig. 4B). Quantitation of ously, mature bantam miRNA was mostly associated with AGO1, relative incorporation of the various siRNAs and miRNAs into

A BCß-elim dsRNA GFP dcr-1 dcr-2 ago1 ago2 Input IP AGO1IP Flag-AGO2IP myc - +

si-FHV RNA2 si-FHV RNA2 si-FHV RNA2 (- strand) (- strand) si-FHV RNA2 hp-CG4068B si-FHV RNA2 (+ strand) (+ strand)

hp-CG4068B bantam hp-CG4068B

2S rRNA bantam bantam

Fig. 4. Biogenesis and terminal properties of FHV-derived siRNAs. (A) Accumulation of FHV siRNAs after depletion of components of the miRNA/RNAi pathways. Blotting for the bantam miRNA and hp-CG4068B endo-siRNA indicate loss of mature miRNA after dcr-1 or ago1 depletion, and loss of mature siRNA after dcr-2 or ago2 depletion, as expected. In contrast to hp-CG4068B, FHV-derived siRNAs are lost only after depletion of dcr-2, and not with ago2; these were detected with a mixture of sense and antisense FHV probes. (B) Coimmunoprecipitation of small RNAs with AGO1 or Flag-AGO2. Bantam miRNA is predominantly associated with AGO1, whereas hp-CG4068B endo-siRNA is predominantly associated with AGO2. FHV siRNAs that are loaded in effector complex associated primarily with AGO2, but most of the viral siRNAs did not associate with either AGO protein. Sense and antisense FHV siRNAs were detected separately in these experiments; input lane ϭ 25% of input used for IP. These data are quantified in Fig. S3.(C) Terminal nucleotide properties of small RNAs. Endo-siRNAs are resistant to ␤-elimination, whereas miRNAs are sensitive and migrate more quickly. FHV-RNA2 siRNAs are similarly sensitive to ␤-elimination.

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813412106 Flynt et al. Downloaded by guest on September 25, 2021 AGO1 and AGO2 are provided in Fig. S3. Therefore, although Strategy of Virus Defense and Counterdefense. Latent infection of there is indeed preferred sorting of viral siRNAs to AGO2, the FHV, a single stranded RNA virus, was associated with the majority of them are denied entry into AGO proteins. relatively equivalent accumulation of siRNAs from positive and Of Drosophila small regulatory RNAs in the 21–24 nt pool, only negative strands. Because Ϫ strand transcripts usually account for those that are incorporated into AGO2 silencing complex are only 1–5% of corresponding ϩ strands, these siRNA signatures subject to 3Ј modification by the Hen1 methyltransferase; bulk indicate that the double stranded FHV replication intermediate is miRNAs are unmethylated (13). Unmethylated RNAs are sensitive the substrate for Dicer-2-mediated defense. However, the entirety to periodate oxidation and ␤-elimination at the terminal nucleotide, of the FHV replication intermediate was not equally susceptible to and consequently migrate as lower molecular weight species (13, 26, dicing. Instead, the punctuated spatial distribution of FHV-derived 27). As shown previously, most of the mature hp-CG4068B was siRNAs suggested these as entry locations that are especially resistant to ␤-elimination (18, 24), reflecting its efficient incorpo- sensitive to dicing. Similar findings were recently reported by Ding ration into AGO2 complex (Fig. 4C). Almost all mature bantam and colleagues using an acute FHV infection system (5). was sensitive to ␤-elimination, reflecting the efficacy of this treat- The recent report that FHV replication is spatially restricted to ment and the fact that very little of this miRNA is sorted to AGO2. spherules within the mitochondrion offers a picture in which the This assay revealed that bulk viral siRNAs from both the ϩ and Ϫ host RNAi machinery may have limited access to replicating virus strands were similarly sensitive to ␤-elimination, confirming their (21). FHV encodes a double-stranded RNA binding protein (B2) inefficient loading into AGO2 complexes (Fig. 4C). Altogether, that protects it from the host RNAi response (1, 2, 19, 20). We these data demonstrate that although the RNAi pathway maintains showed that ϩ and Ϫ strands of FHV are associated with B2. These FHV latency in S2 cells, this is poorly correlated with the activity data suggest that B2 protects FHV by binding the Dicer-2- of bulk AGO2-loaded viral siRNAs as transacting inhibitory susceptible replication intermediate, as opposed to an intramolecu- species. larly double-stranded region of the coding viral genome (see also ref. 5). In light of these spatial constraints, future studies of virus Discussion defense will need to address cell biological aspects of virus recog- Role of Dicing During Maintenance of Drosophila Virus Latency. Here, nition and evasion. This seems reminiscent of the recent appreci- we have examined how latency of FHV in persistently infected cells ation of the cell biological constraints that compartmentalize is maintained by the RNAi pathway. Viral siRNAs were abundant miRNA-mediated repression within the cell (31).

in small RNA libraries, and shown by Northern blot analysis to be GENETICS produced in a Dicer-2-dependent fashion. Unexpectedly, our data Implications of Latent Viral siRNA Biogenesis for Small RNA Sorting. reveal that bulk viral siRNAs in these cells do not program active We showed that FHV-derived siRNAs were preferentially incor- effector complexes. These siRNAs fail to repress complementary porated into AGO2 relative to AGO1, consistent with recent sensor transcripts, associate with AGO2 rather weakly, and are sequencing data from Argonaute immunoprecipitates from latently sensitive to ␤-elimination. Taken together, our data suggest that in infected cells (17). However, the bulk of siRNAs were not loaded, addition to the well-appreciated role for dicing to generate siRNAs and were not 3Ј modified as expected for AGO2-loaded small that cleave viral transcripts, the direct dicing of double stranded RNAs (13, 14). Similarly, Ding and colleagues recently detected a replication intermediates appears to play an appreciable role in pool of FHV-derived siRNAs generated from an attenuated form maintaining FHV in a latent state. of FHV lacking the B2 suppressor of RNAi were not loaded into Although the relative contributions of antiviral dicing and slicing either AGO1 or AGO2 effector complexes, and lacked a 3Ј remain to be fully elucidated, it is worth considering whether there modification (5). Moreover, the Ding laboratory showed that viral might be distinct phases and activities of the RNAi pathway during siRNAs generated during an acute infection also did not depend on antiviral response. Upon onset of active infection, siRNAs are both AGO2 for their accumulation; in fact they observed over- generated and used to target coding RNAs, causing destruction of accumulation of FHV-derived siRNAs after infection of ago2 viral RNAs. However, if the infection can be adequately controlled, mutants with attenuated FHV (5). The accumulation of unloaded the necessity to sort viral siRNAs into RISC might conceivably viral siRNAs during acute and latent infections contrasts strongly become less critical, whereas Dicer-2 remains active in turning over with the behavior of diverse classes of endo-siRNAs, for which the remaining persistent viral RNAs. Dicer-2 was also recently Northern analysis demonstrates them to require AGO2 for their shown to induce the antiviral Vago protein, apparently in an stable accumulation (17, 18, 23, 24, 32). AGO2-independent manner, demonstrating that the role of Why can the generation and loading of viral siRNAs, unlike that Dicer-2 extends beyond simply the generation of antiviral RISC of endo-siRNAs, be uncoupled? In principle, the abundant pro- (28). Nevertheless, there is clearly a role for AGO2 in suppressing duction of virus during acute infection might saturate the loading latent FHV (17), as with acute FHV infection (2, 5, 19). It is possible machinery, potentially yielding a population of unloaded siRNAs. that Dicer-2 requires its effector AGO2 to efficiently clear siRNA However, the pool of viral siRNAs produced during latent infection duplexes. Alternatively, it might be that a very small amount of is perhaps Ͻ5% the pool of endo-siRNAs that are loaded into AGO2 programmed with viral siRNA suffices to maintain FHV AGO2, indicating that the failure to load latent viral siRNAs is not latency. a consequence of saturating the loading machinery. Indeed, the A caveat of our studies is that a pool of latently infected virus may klarsicht locus alone accounts for Ϸ16% of the contents of AGO2 have undergone genomic rearrangement, which might alter its in S2-NP and S2-GMR cells (17, 23), demonstrating that the highly properties. Previous work described the spontaneous appearance of abundant production of siRNAs from a single locus is possible. attenuated, internally deleted FHV isoforms (29, 30), whose de- Instead, these observations might suggest the existence of licensing struction by Dicer-2 might account for our ability to detect siRNAs measures that select appropriate siRNAs for loading and/or reten- via sequencing and Northern blot analysis. However, this scenario tion in effector complex. does not account for the robust derepression of full-length viral Indeed, mechanisms for siRNA selection were hinted at by recent RNA accumulation upon the depletion of dcr-2. In addition, we studies of Drosophila endo-siRNAs. We and others recently char- demonstrated that latent FHV retains most of its function and can acterized hairpin RNAs, which are long inverted repeat transcripts readily infect and kill naïve cells. Future studies on the dynamics and that are processed by Dicer-2 into siRNAs that preferentially load kinetics of de novo infection by previously latent virus may help to AGO2 (12). Although the small RNA cloning patterns from some establish the parameters of successful establishment of the latent hpRNAs revealed signature evidence for the processive action of state, and address whether there might be shifting influences of Dicer-2 to yield phased siRNA duplexes (17, 18, 24), some hpRNAs viral dicing and slicing during this transition. generated abundant siRNAs from isolated regions embedded in the

Flynt et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 25, 2021 midst of long double-stranded precursors (24). Such patterns AATCAGCTTTCAAAATGATCTCA-3Ј). A DNA probe complementary to 2S rRNA suggest that bulk siRNAs generated by dicing across a long sub- (5Ј-TACAACCCTCAACCATATGTAGTCCAAGCA-3Ј) was used to determine equal strate were not loaded into effector complex, and were instead loading. discarded. In at least some systems, siRNA-bearing AGO proteins To analyze B2-associated RNAs, we cotransfected UAS-6xHis-3xMyc-FHV-B2 (35) with Ub-Gal4 into S2Rϩ cells. After 48 h cells were lysed in 20 mM Tris (pH have been shown to be limiting for siRNA accumulation (33). 7.4), 200 mM NaCl, 2.5 mM MgCl2, 0.05% Nonidet P-40 with Complete Pro- Consequently, licensing mechanisms for siRNA loading might be tease inhibitors (Roche). Immunoprecipitation was carried out with mouse important to ensure the optimal occupancy of AGO proteins with anti-6xHis or mouse anti-Myc (Santa Cruz Biotechnology), and T7 antibodies the desired siRNAs. In principle, an improved understanding such (Novagen) bound to GammaBind G Sepharose (GE Healthcare). RNAs were siRNA selection mechanisms will improve the ability to design isolated from beads by phenol choloform extraction followed by ethanol effective artificial siRNAs for designed purposes. precipitation. RNAs were separated on a 6% Urea acrylamide gel and blotted onto nylon membranes (Genescreen). 32P end-labeled DNA probes were used ϩ Materials and Methods to detect the 3Јend of the FHV RNA2 strand (5Ј-ACCTTAGTCTGTTGACTTA- AACTGTTTGGG-3Ј) or the 3Ј end of the FHV RNA2 Ϫ strand (5Ј-GTAAACAAT- Library Construction and Analysis. Independent 50-␮g samples of total RNA from TCCAAGTTCCAAAATGGTCAAC-3Ј). S2Rϩ cells or S2-GMR were fractionated on polyacrylamide gels, and the Ϸ18–28 Immunoprecipitation of AGO complexed RNAs was carried out in cells stably nt fraction was cloned according to the protocol of Hannon (17). Each library was transfected with Flag-HA-tagged AGO2 (17). These cells were lysed in 30 mM subjected to a single lane of sequencing using the Illumina platform. The reads Hepes-KOH (pH 7.3), 150 mM K acetate, 2 mM Mg acetate, 0.1% Nonidet P-40, were clipped of the 3Ј linkers, and submitted to the National Center for Biotech- and 5 mM DTT with Complete Roche Protease inhibitors. Flag-HA-AGO2 was nology Information Gene Expression Omnibus under the accessions GSM343832 immunoprecipitated using Anti-Flag M2-Agarose (Sigma). Rabbit anti-AGO1 and GSM343833 (S2Rϩ, first and second biological replicates) and GSM361908 (Abcam) and mouse anti-myc (Santa Cruz Biotechnology) were immobilized on (S2-GMR) cells. We also used data from an independent biological replicate of GammaBind G Sepharose (GE Healthcare) before immunoprecipitation. RNAs S2-GMR reads that we reported in GSM272652 (32). were extracted as described above and analyzed on 15% Urea acrylamide gel and Using Flock House Virus genomic sequences obtained from National Center Northern blotting. for Biotechnology Information, we extracted Ն18 nt reads that mapped perfectly to the viruses for analyses of the size and spatial distribution of viral small RNAs. Luciferase Assays. Luciferase assays were performed as described in ref. 34 using To assemble the FHV genome from small RNA reads, we also considered Ն18 nt dual Renilla/Firefly sensors and the Dual-Glo luciferase kit (Promega). Three reads with 1 or 2 mismatches/1 nt deletion/1 nt insertion to the reference FHV different 70 nt regions of the ϩ or Ϫ strand of FHV RNA2 were cloned into a sequence, discarding those reads that also mapped perfectly to the dm3 assembly modified version of psiCheck2 (Promega) (34): 169–239 bp (region1), 433–503 bp of the D. melanogaster genome. (region 2), and 530–599 bp (region 3). Mutant controls corresponding to each segment were made with changes at every 4th base and similarly cloned into 32 RNA Molecular Biology. A mixture of 4 P-labeled DNA probes were used to psiCheck2. Values from wild-type sensors were normalized to mutant controls to detect FHV RNA1, RNA2 and RNA3 genomic RNAs by agarose Northern blot determine the relative repression. This experiment was performed in 3 cell lines, ϩ analysis. Two probes were complementary to the shared strand of RNA1 and S2Rϩ, S2-NP, and S2. RNA3 (5Ј-ACCTCTGCCCTTTCGGGCTAGAAC-3Ј and 5Ј-TCACTTCCGGTTGTTG- Ј ϩ GAAGGC-3 ), whereas the other two were complementary to the strand of ACKNOWLEDGMENTS. We thank Gregory Hannon (Cold Spring Harbor Labo- Ј Ј Ј RNA2 (5 -AGGAGGACACTTGATCGGATCTGG-3 and 5 -GGGATCGGTGTT- ratory, Cold Spring Harbor, NY) for Flag-HA-AGO2 ; Norbert Perrimon GAAGTCAGGTG-3Ј). A DNA probe complementary to U6 (5Ј-GCAGGGGCCAT- (Harvard Medical School, Boston, MA), Ram Dasgupta (New York University, New GCTAATCTTC-3Ј) was used to determine relative loading. York), Gerald Rubin (Janelia Farm, Ashburn, VA), Philip Zamore (University of ␤-elimination and detection of small RNAs by acrylamide Northern blot anal- Massachusetts Medical School, Worcester, MA), Lucy Cherbas (Indiana University, ysis was performed as described (13, 34). We used 4 LNA probes that are com- Bloomington, IN), and Sue Celniker (Lawrence Berkeley National Laboratory, plementary to FHV RNA2 ϩ strand (5Ј-GGGAAAGCGCCGCCATATTCATGCC-3Ј Berkeley, CA) for Drosophila cell cultures and/or RNA samples. Illumina sequenc- Ј Ј Ϫ Ј ing was performed by the British Columbia Genome Science Centre (Vancouver). and 5 -GTGCGAAGGCACACTTGAGAAACGC-3 ) or FHV RNA2 strand (5 - Katsutomo Okamura performed preliminary experiments and provided helpful Ј Ј GCGTTTCTCAAGTGTGCCTTCGCAC-3 and 5 -GCATGAATATGGCGGCGCTTTC- discussion. This work was supported by the Sidney Kimmel Cancer Foundation, CCG-3Ј). hp-CG4068B was detected by an LNA probe (5Ј-GGAGCGAACTTG- the Alfred Bressler Scholars Fund and National Institutes of Health Grants R01- TTGGAGTCAA-3Ј), and bantam was detected by a DNA probe (5Ј- GM083300 and U01-HG004261.

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