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Dicing of Viral Replication Intermediates During Silencing of Latent Drosophila Viruses

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Dicing of Viral Replication Intermediates During Silencing of Latent Drosophila Viruses Dicing of viral replication intermediates during silencing of latent Drosophila viruses 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 Cancer 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 infection in plants, nematodes bipartite FHV genome. Unexpectedly, the abundant siRNAs de- and flies. However, little is known about how RNAi combats rived from latent virus were not effective in silencing reporters viruses during persistent or latent infections. 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 cell 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 capsid proteins. Ginterference (RNAi) pathway generate viral small interfering Although productive FHV infection induces apoptosis 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 enzymes 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.
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