Drosha As an Interferon-Independent Antiviral Factor

Drosha as an interferon-independent antiviral factor

Jillian S. Shapiroa,b,1, Sonja Schmida,1, Lauren C. Aguadoa,b, Leah R. Sabinc, Ari Yasunagac, Jaehee V. Shima, David Sachsd, Sara Cherryc, and Benjamin R. tenOevera,b,2

aDepartment of Microbiology, bIcahn Graduate School of Biomedical Sciences, and dDepartment of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and cDepartment of Microbiology, University of Pennsylvania, Philadelphia, PA 19104

Edited by Bryan R. Cullen, Duke University, Durham, NC, and accepted by the Editorial Board April 3, 2014 (received for review October 17, 2013) Utilization of antiviral small interfering RNAs is thought to be largely of hundreds of IFN-stimulated genes (ISGs) through a ubiquitous restricted to plants, nematodes, and arthropods. In an effort to de- IFN-I receptor (encoded by the Ifnar1 gene) (18). termine whether a physiological interplay exists between the host Despite the lack of robust vsiRNA production, chordates have small RNA machinery and the cellular response to virus infection in retained genome-encoded microRNAs (miRNAs). These non- mammals, we evaluated antiviral activity in the presence and ab- coding RNAs are transcribed by RNA polymerase II and processed sence of Dicer or Drosha, the RNase III nucleases responsible for in a stepwise fashion by two RNase III enzymes: first, Drosha in the generating small RNAs. Although loss of Dicer did not compromise nucleus; and second, Dicer in the cytoplasm (20–26). Similar to the cellular response to virus infection, Drosha deletion resulted in vsiRNAs, miRNAs are also capable of exerting RNAi although they a significant increase in virus levels. Here, we demonstrate that more commonly act to fine-tune host gene expression through diverse RNA viruses trigger exportin 1 (XPO1/CRM1)-dependent Dro- translational repression and/or mRNA deadenylation and are sha translocation into the cytoplasm in a manner independent of de thought to contribute to cellular fitness (27–32). Given the modest novo protein synthesis or the canonical type I IFN system. Addition- repression of miRNAs on their targets, a property that results from ally, increased virus infection in the absence of Drosha was not due to imperfect binding complementarity, they are unlikely to serve as a loss of viral small RNAs but, instead, correlated with cleavage of direct inhibitors of viral transcripts (33). However, viruses can be viral genomic RNA and modulation of the host transcriptome. Taken engineered to encode perfect complementary target sites for en- together, we propose that Drosha represents a unique and conserved dogenous miRNAs as an effective mechanism to attenuate virus arm of the cellular defenses used to combat virus infection. replication (34–41).

Despite the apparent evolutionary loss of vsiRNAs as an an- MICROBIOLOGY RNAi | microRNA | miRNA | Rnasen | innate immunity tiviral defense in chordates, there are many overlaps between the RNAi and IFN-I pathways, most notable being that both IFN-I n plants, nematodes, and arthropods, a major response to virus and RNAi can be triggered by the presence of dsRNA (42, 43). Iinfection is Dicer-dependent generation of virus-derived small Furthermore, a number of proteins involved in miRNA pro- interfering RNAs (vsiRNAs) (1, 2). vsiRNAs associate with the duction have also been implicated in the IFN-I response. For RNA-induced silencing complex (RISC) and mediate cleavage example, the dsRNA-binding proteins TRBP and PACT, which of homologous viral RNA, attenuating virus replication in a aid in precursor-miRNA dicing, RISC maturation, and target process termed antiviral RNA interference (RNAi) (3, 4). Al- silencing, have also been reported to inhibit and activate effec- though many components of antiviral RNAi are conserved in tors of the IFN-I pathway, respectively (44, 45). In addition, both – chordates, the small RNA-mediated response to virus infection the ubiquitous and IFN-I inducible isoform of ADAR1 can has largely been replaced with the protein-based type I IFN function to alter miRNA expression (46) and associate with Dicer (IFN-I) response although evidence for mammalian RNAi has to enhance enzyme activity (47). Conversely, many viruses in- teract with Drosha and Dicer for the production of viral miRNAs recently been reported in some cell types against particular or to regulate the levels of viral transcripts (48–51). The range in viruses (5–7). Indeed, ectopic expression of siRNAs directed interplay between virus and the mammalian miRNA pathway against viral genomes in diverse cell types potently inhibits virus – demonstrates the capacity for cross-talk between these two sys- replication of a wide range of viruses (8 15). However, vsiRNAs tems, but the physiological relevance of this cross-talk remains have been difficult to detect in IFN-I–sensitive cells (2, 16, 17). These data suggest that, whereas chordates may not produce Significance robust levels of vsiRNAs, they are capable of harnessing the small RNA machinery in an antiviral capacity when presented Virus infections must be combated at a cellular level. The strat- with the proper substrate. These same data also suggest that egies used to inhibit virus differ dramatically when comparing mammalian RNA viruses have not incurred any clear selective plants and insects to mammals. Here, we identify an evolution- pressure to inhibit small RNA-mediated signaling, in contrast ary conserved antiviral response that is independent of these to the IFN-I induction pathway where virus antagonism is known defenses. We demonstrate that an RNA nuclease called common (18). Drosha is repurposed during virus infection to cleave viral RNA In mammals, RNA virus infection is recognized in response to and modulate the cellular environment as a means of inhibiting replication, as this process generates a diverse array of pathogen virus replication. associated molecular patterns (PAMPs). PAMPs include double stranded RNA (dsRNA), RNA with an exposed 5′ triphosphate, Author contributions: J.S.S., S.S., L.R.S., and B.R.t. designed research; J.S.S., S.S., L.C.A., or RNA lacking a 2′ O-methyl–containing cap (18). In the vast L.R.S., A.Y., and J.V.S. performed research; S.C. contributed new reagents/analytic tools; J.S.S., S.S., L.C.A., L.R.S., A.Y., J.V.S., D.S., and B.R.t. analyzed data; and J.S.S., S.S., L.R.S., majority of cells, PAMPs are detected by one of the two PAMP S.C., and B.R.t. wrote the paper. recognition receptors (PRRs): RIG-I (Encoded by the Ddx58 The authors declare no conflict of interest. gene) and MDA5 (18). PRR detection culminates in a signal This article is a PNAS Direct Submission. B.R.C. is a guest editor invited by the Editorial transduction event that includes activation of the IFN regulatory Board. factors (IRFs) by tank-binding kinase 1 (TBK1) (19). Kinase ac- 1J.S.S. and S.S. contributed equally to this work. tivation results in assembly of a multisubunit enhancer that pro- 2To whom correspondence should be addressed. E-mail: [email protected]. motes transcription of the IFN beta gene, a member of the IFN-I This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. family. IFN-I production subsequently results in the up-regulation 1073/pnas.1319635111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1319635111 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 poorly understood. Supporting data for direct RNAi against viral biogenesis throughout the course of the Drosophila cell infection RNAs in mammalian cells includes evidence for RNase III-like (Fig. 1B). Next, to determine whether this activity was also Dro- activity in the restriction of retrotransposons and two RNA virus sha-dependent, we depleted cells of this nuclease and assessed infection models (5, 6, 52). Given these findings and associations, cytoplasmic (c)-pri-miR-124 processing (Fig. 1C and Fig. S1). we sought to determine whether Dicer or Drosha, the only mam- Drosha depletion abrogated the ability of SINV124 to produce malian RNase III nucleases, contributed to the mammalian re- a mature miRNA and resulted in an enhancement in the level of sponse to virus infection in somatic cells, which are the major unprocessed virus-derived pri-miR-124 and viral RNA (Fig. 1C targets of viral infection. and Fig. S1). Taken together, these data implicate a possible role for Drosha in the cleavage of the cytoplasmic virus-derived RNA Results transcripts in Drosophila and suggest increased virus replication in Drosha Translocation Is a General Response to RNA Virus Infections. the absence of the nuclease. Recent evidence has demonstrated the capacity to engineer cytoplasmic viruses to produce miRNAs (53–57). Subsequently, we Sindbis Virus Is Susceptible to an RNAi-Mediated Antiviral Response. found that cytoplasmic miRNA synthesis was dependent on a Given the broad accumulation of cDrosha in response to a panel Drosha translocation event to process the miRNA from Sindbis of RNA viruses, we hypothesized that Drosha may play a role in virus (SINV) (58). Given the recent findings relating to the an RNAi-like response perhaps related to the recent findings in – ability of the miRNA machinery to naturally exert an antiviral mammalian cells (5 7). We hypothesized that, if Drosha had a response in mammalian fibroblasts (6), we sought to investigate role in antiviral RNAi, then both Drosha and Dicer would have whether the SINV-induced translocation of Drosha into the antiviral properties where products from Drosha would be fed to cytoplasm represents a broad antiviral response. Therefore, we Dicer, which would then produce substrates for RISC that would investigated Drosha localization in response to infection with silence viral RNAs. To determine whether these two RNase III a positive sense virus (SINV), a negative sense virus [vesicular nucleases, Drosha and Dicer, contribute to the cellular response stomatitis virus (VSV)], and a nuclear, segmented RNA virus to virus infection, we measured the impact of virus replication in [mutated influenza A virus (mIAV)], which lacks its main an- the presence or absence of each nuclease. We chose to further tagonist of the antiviral response [nonstructural protein (NS1), study SINV as it is a virus model that has previously been shown described in ref. 59], and in response to treatment with the ca- to be capable of generating miRNAs, suggesting that it does not disrupt the host machinery responsible for small RNA biogenesis nonical viral PAMP, dsRNA (Fig. 1A). Interestingly, we found (53). However, to first ensure that the virus lacked a suppressor that, despite exclusive expression of Drosha in the nucleus in of RNA silencing (SRS), we assessed whether small RNAs could mock-treated cells, there was robust translocation to the cyto- be harnessed to inhibit SINV replication by engineering the virus plasm in response to SINV, VSV, mIAV, or dsRNA (Fig. 1A). with a scrambled RNA (scbl) in its 3′ UTR or two or four Furthermore, cytoplasmic Drosha (cDrosha) was evident during miR-124 target sites in the same location (2 × 124T, or 4 × 124T, the early hours of infection and dsRNA treatment (Fig. 1A). respectively). Because miR-124 is restricted to neurons, infection These data suggest that detection of a broad range of viral PAMPs of SINV scbl, 2 × 124T, or 4 × 124T in fibroblasts resulted in results in the accumulation of Drosha in the cytoplasm. equal levels of SINV replication as measured by capsid protein synthesis (Fig. 2A). In contrast, exogenous expression of miR-124 Drosha-Dependent Cytoplasmic miRNA Processing Is Conserved in resulted in a complete loss of capsid expression in 2 × 124T or Arthropods. Given the generality of virus-induced Drosha trans- 4 × 124T virus while having no impact on SINV scbl or on host location, we next assessed whether insects also display cDrosha protein disulfide isomerase (PDI) (Fig. 2A). Taken together, activity by assaying miRNA production from a Drosha-dependent, these results suggest that SINV is capable of being targeted by cytoplasmic RNA transcript. Drosophila melanogaster cells are RNAi during infection. permissive hosts of many alphaviruses and, as in mammalian cells, support a cytoplasmic SINV replication cycle (60). As such, Loss of Drosha Results in an Increase in RNA Virus Replication. Given Drosophila cells (DL1) were infected with a recombinant SINV the lack of a SINV SRS, we next used conditional knockout encoding primary (pri)-miR-124 (SINV124) (53), and miR-124 fibroblasts for Drosha or Dicer (Rnasenf/f and Dicer1f/f, respectively) synthesis from the cytoplasmic transcript was monitored. Similar and disrupted each gene using replication-incompetent Adeno- to mammalian infections, SINV124 resulted in miRNA based vectors (AdV) expressing GFP or a GFP-Cre fusion protein (Fig. 2B). Cells were incubated for 5 d to allow for efficient clearance of both the vector and targeted host protein. Small RNA Northern blot analysis confirmed loss of endogenous, Drosha- and Dicer-dependent miR-93, demonstrating loss-of-functional enzymatic activity of both genes (Fig. 2B). In contrast, U6, a small nuclear RNA that does not depend upon Drosha or Dicer, was not impacted by these treatments (Fig. 2B). Drosha-depleted cells were subsequently infected with SINV at a multiplicity of infection (MOI) of 0.1 for a multicycle growth curve and compared against control cells. Interestingly, SINV titers reached significantly higher levels in the absence of Drosha throughout the course of infection (Fig. 2C). SINV capsidproteinalsoaccumulatedtohigherquantitiesinDrosha- Fig. 1. Broad accumulation of cDrosha in virus-infected cells. (A) Immuno- depleted cells, consistent with elevated levels of virus replica- histochemistry of murine fibroblasts infected with SINV (MOI = 3), VSV tion (Fig. 2D). In contrast, depletion of Dicer did not result in (MOI = 1), or mIAV lacking the main viral IFN antagonist nonstructural = a significant alteration in the SINV titers over the course of protein NS1 (MOI 5), or transfected with poly(I:C) for 6 h and stained for infection compared with control cells (Fig. 2E). Western blot nuclei, viral proteins, or Drosha. (B) Small RNA Northern blot of Drosophila cells (DL1) mock-treated or infected (MOI = 1) with SINV WT or with SINV124 also revealed unaltered virus levels between Dicer-deficient for the indicated times. (C) Small RNA Northern blot of DL1 cells treated with and control cells (Fig. 2F). Furthermore, VSV also displayed dsRNA against β-galactosidase (bgal) or Drosha and subsequently infected enhanced replication in the absence of Drosha but not Dicer with SINV124. (Fig. S2 A–D). These data demonstrate that Drosha restricts

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1319635111 Shapiro et al. Downloaded by guest on September 24, 2021 the active transport of Drosha from the nucleus into the cytoplasm in a CRM1-dependent fashion. Next, we determined whether de novo translation is required for the accumulation of cDrosha. To this end, we inhibited translation with cyclohexamide (CHX) and subsequently infected with VSV encoding miRNA-124 (VSV124), which has also been demonstrated to generate a functional miRNA from the cytoplasm (55). VSV124, rather than SINV124, was chosen for this assay as SINV requires host translation to generate virus transcripts. In contrast, VSV packages its own RNA-dependent RNA polymerase, and, therefore, CHX treatment does not inhibit pri-miR-124 synthesis although viral protein production is lost (61). We found that the production of VSV-derived miR-124 was not dependent on translation as mature miR-124 accumulated to higher levels compared with mock-treated cells infected with VSV124, despite a complete loss of VSV G protein (Fig. 3D). Taken together, these data demonstrate that RNA virus infection results in the active transport of Drosha from the nucleus into the cytoplasm in a CRM1-dependent fashion. Given our observation that Drosha relocalizes upon infection with diverse viruses and dsRNAs, which are canonical PAMPs that are sensed by RIG-I, we set out to determine whether the relocalization of Drosha was dependent on RIG-I or classical IFN signaling. We chose to analyze SINV124 miRNA processing in the presence and absence of RIG-I as cytoplasmic pri-miRNA (c-pri-miRNA) processing is dependent on cDrosha (Fig. 3C). Surprisingly miR-124 accumulated to WT levels in the absence

− − MICROBIOLOGY of RIG-I (Ddx58 / ), demonstrating that this dsRNA sensor is not required for cDrosha activity (Fig. S3B). Moreover, cDrosha − − Fig. 2. Drosha restricts virus replication. (A) 293T cells transfected with activity was retained in the absence of TBK1 (Tbk1 / )andIFN-I − − empty or miR-124 producing vector for 36 h and subsequently infected with signaling (Ifnar1 / )(Fig. S3 C and D). These data suggest that the SINV expressing a scrambled sequence (scbl) or two or four miR-124 target relocalization of Drosha represents a unique, IFN-I independent sites (2 × 124T or 4 × 124T, respectively) in the 3′ UTR at an MOI of 1 for 24 h. (Top and Middle) Western blot for SINV capsid or PDI. (Bottom) Small RNA arm of the cellular antiviral response. Northern blot of ectopically expressed miR-124. (B) Small RNA Northern blot ’ of Rnasenf/f or Dicer1f/f fibroblasts treated with replication-incompetent Cytoplasmic Translocation Is Necessary for Drosha s Antiviral Activity. Adeno-based vectors (AdV) expressing GFP or a GFP-Cre fusion protein (Cre) It has previously been reported that translocation of Drosha into for 5 d and probed for miR-93 (Upper)orU6(Lower). (C) Plaque assay of the nucleus is dependent upon phosphorylation of serines 300 Rnasenf/f fibroblasts treated with AdV-GFP or AdV-Cre and subsequently and 302 (62). To investigate whether these same residues play infected with recombinant SINV (MOI = 0.1). (D) Western blot for same a role in virus-induced cDrosha, we used GFP-tagged Drosha f/f conditions as in C.(E) Plaque assay of Dicer1 fibroblasts treated with AdV- constructs expressing Alanines (A) or phosphomimetic residues = GFP or AdV-Cre and subsequently infected with recombinant SINV (MOI 0.1). (Aspartate, D, or Glutamate, E) at these positions. Consistent (F) Western blot for same conditions as in E.DatainC and E are represented as the mean ± SEM for n = 3. *Significant P value of <0.05, using a two-tailed, with previous reports, transfection of GFP-Drosha (WT), unpaired Student’s t test. S300/302A (2A), or S300E/S302D (ED) demonstrated that these residues are critical components in defining the cellular localization of Drosha: WT and ED were nuclear whereas 2A was pre- RNA virus replication whereas loss of Dicer (and consequently dominantly cytoplasmic (Fig. 4A). Thirty-six hours posttransfection miRNAs) does not significantly contribute to the mammalian response to virus infection, at least in the context of primary fibroblasts.

Virus Infection Results in Nuclear Export of Drosha. We next sought to define the mechanism responsible for the accumulation of Drosha in the cytoplasm upon SINV infection because cDrosha may account for the antiviral activity. To discern whether cDrosha was the product of active nuclear export or was the result of newly synthesized protein retention, we investigated the requirement for the nuclear export protein CRM1 in cytoplasmic processing of primary miRNAs (pri-miRNAs). Subsequent to RNAi-mediated depletion of CRM1, cells were infected with SINV124, and subcellular fractionation was performed (Fig. 3 A and B). Infection with SINV124 resulted in accumulation of Drosha in the cyto- Fig. 3. Host requirements for Drosha export. (A) Western blot of 293Ts at 48 plasm in a CRM1-dependent fashion (Fig. 3B and Fig. S3A). hpt with 50 nM control (ctrl) pool of siRNA or siRNA pool directed against Excitingly, CRM1-depleted cells were no longer capable of sup- Crm1.(B) Western blot of subcellular fractionation of conditions in A additionally mock-treated or infected with SINV124 (MOI = 3, 8 hpi). (C) Small RNA porting miRNA biogenesis from the cytoplasmic SINV-derived Northern blot of conditions in A additionally mock-treated or infected with pri-miRNA whereas endogenous miR-93 was not impacted (Fig. SINV124 (MOI = 3, 16 hpi). (D) MEFs mock-treated or treated with CHX for 2 h 3C). These data demonstrate that RNA virus infection results in and subsequently mock-treated or infected with VSV124 (MOI = 1, 8 hpi).

Shapiro et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 (Fig. 5A). We next probed for small RNAs specific to SINV gRNA using a probe that bound to a sequence in the ORF of the nonstructural polyprotein of SINV. Indeed, we found that Drosha mediated cleavage of the viral gRNA to yield an ∼55-nt product (Fig. 5A). Furthermore, we observed an increase in RNAs of ∼20– 25 nt. These data suggest that Drosha is capable of cleaving the viral gRNA, likely at secondary structures, which may then result in RNA degradation because the 5′ and 3′ would not be protected via a cap or poly(A). Given the ability of Drosha to cleave structures embedded in the viral gRNA (Fig. 5A), we investigated whether Drosha impacted the small RNA profile during virus infection. Small RNA deep sequencing of SINV-infected fibroblasts captured over 675,000 reads with coverage over the complete SINV genome (Fig. S4A and Dataset S1). However, in contrast to what was reported for a mutant Nodavirus (5, 6), we found no enrichment in 21-nt RNAs characteristic of vsiRNAs (Fig. S4B). This is in agreement with Fig. 4. Phosphorylation requirements for antiviral Drosha activity. (A)Im- other recent studies performed in fibroblasts (64). Lack of ca- munohistochemistry of 293T cells transfected with the indicated plasmids nonical vsiRNA production prompted us to investigate the possi- and subsequently mock-treated or infected with SINV (MOI = 5, 4 hpi) at 36 hpt. Cells were stained for SINV capsid, and GFP was imaged. (B) Plaque bility that virus stem loops are targets for the dsRNA nuclease assay of supernatants from BHK cells, cotransfected with the indicated activity of Drosha in its capacity to limit virus replication in the plasmids and with SINV genomic RNA (gRNA) for 24 h. Data are represented cytoplasm. To this end, we cloned small RNAs (19-25 nt) from as the mean ± SD for n = 3. *Significant P value of <0.05, using a two-tailed, wild-type and Drosha-deleted cells infected with SINV (Fig. 5B and unpaired Student’s t test; ns, nonsignificant P value of >0.05. (C) Western blot for the same conditions as B.

(hpt) of the Drosha constructs, SINV infections were performed at an MOI of five, and GFPNLS and GFP-Drosha constructs were visualized 4 h postinfection (hpi). Although SINV infection had no impact on the cellular localization of the control protein, GFPNLS, infection resulted in cytoplasmic accumulation of GFP-Drosha-WT (Fig. 4A), as observed with endogenous Drosha (Fig. 1A)(58). Furthermore, virus infection did not impact the constitutive cytoplasmic localization of the 2A mutant. In contrast, GFP-Drosha- ED failed to translocate to the cytoplasm in response to virus infection (Fig. 4A). These results suggest that serine 300 and 302 must be dephosphorylated in virus-infected cells to allow for the cytoplasmic accumulation of Drosha. Next, we determined whether cytoplasmic localization was required for Drosha’s antiviral activity. We expressed GFP or the GFP-Drosha constructs (wt, 2A, or ED) and transfected infectious SINV genomic RNA (gRNA) (Fig. 4B). A plaque assay was used to assess virus replication. These studies found that the overexpression of WT or ED forms of Drosha led to a one log attenuation of infection whereas Drosha 2A potently inhibited SINV replication by more than two logs compared with GFP (Fig. 4B). These results were also observed at the protein level as measured by immunoblot (Fig. 4C). These data suggest that Drosha’s antiviral activity is potently exerted in the cytoplasm and that Drosha levels are limiting because ectopic expression is restrictive. Furthermore, these data suggest that there is a virus- inducible phosphatase that drives the relocalization of Drosha during virus infection and that this relocalization is required for Drosha to elicit its full antiviral activity. Drosha-mediated cleavage of viral RNA. To parse out Drosha’s nuclear and cytoplasmic mechanisms of antiviral activity, we in- Fig. 5. Drosha impacts viral and host RNAs during infection. (A) In vitro vestigated whether cDrosha was capable of processing the cleavage reaction of SINV124 genome incubated with purified Flag-tagged cytoplasmic SINV genome to generate 21-nt vsiRNAs recently GFP or Drosha and probed for miR-124 (124, Left) or SINV gRNA (SINV, Right). described in fibroblasts (5–7). To this end, we performed an in (B) Small RNA deep sequencing of Rnasenf/f fibroblasts treated with AdV-GFP vitro cleavage assay on SINV gRNA derived from SINV124 as or AdV-Cre for 5 d and subsequently infected with SINV (MOI = 3, 8 hpi). (C) previously described (63). Purified Flag-tagged GFP or Drosha Small RNA deep sequencing of Drosophila DL1 cells treated with indicated = was used to perform in vitro cleavage assays that were subsequently dsRNA for 3 d and subsequently infected with SINV (MOI 20, 96 hpi). (D) Heat map of RNA-seq reads for Rnasenf/f fibroblasts treated with AdV-GFP or AdV- analyzed by small RNA Northern blot. As a control, we assessed = ’ Cre for 5 d and subsequently mock-treated or infected (SINV, MOI 3, 8 hpi). Drosha s ability to cleave the pri-miR-124 embedded in the SINV Data represent the relative fold gene expression of AdV-Cre–treated, SINV- gRNA. Not surprisingly, we found that Drosha is able to specifi- infected cells over AdV-GFP–treated, SINV-infected cells and is graphed as fold cally cleave the viral gRNA to produce an ∼55-nt preliminary change over mock-treated cells. Only mRNA transcripts with a greater than (pre)-miR-124 whereas purified GFP did not process this miRNA fivefold increase or decrease are represented.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1319635111 Shapiro et al. Downloaded by guest on September 24, 2021 Dataset S2). Although loss of Drosha resulted in a significant en- replication (Fig. 5). In addition, mRNA seq data from infected cells hancement in overall small RNA reads, no change in the profile or with or without Drosha demonstrated Drosha-dependent changes genomic positioning of RNAs could be detected. Furthermore, we in the host transcriptome that, in concert with the cleavage of viral analyzed the small RNA profiles in Drosophila cells infected with RNA, likely contribute to its antiviral property. SINV in the presence or absence of Drosha. Again, we observed Recent data suggest that mammalian cells retain some aspect of that Drosha depletion increased the number of virus-specific the antiviral RNAi response (5, 6). We indeed identified small reads without changing the overall profile or genomic locations RNA fragments from in vitro Drosha cleavage assays, which may (Fig. 5C and Dataset S3). This observation is in agreement with have correlates with vsiRNAs in fibroblasts. However, the size independent published fly data that found that the loss of Drosha distribution of these small RNAs was not representative of ca- resulted in elevated levels of vaccinia virus, Drosophila C virus, nonical siRNAs. Alternatively, the previously described mam- and VSV RNA (2). These data suggest that cDrosha does not malian Dicer-dependent siRNAs may be produced downstream mediate its antiviral activity through generation of vsiRNAs. of Drosha processing. Because we found no role for Dicer in our Drosha modulates the transcriptome during virus infection. In addition assays, the role for Drosha in antiviral defense described here is to cleavage of viral RNA, we additionally assessed the Drosha- distinct from previous observations. Indeed, we speculate that the dependent changes in gene expression upon virus infection. We antiviral activity imposed by Drosha is rather a consequence of (i) performed RNA-Seq analysis, which demonstrated that Drosha the nuclease’s altered cellular localization, (ii) cleavage of novel influenced the mRNA profile of cells in the absence of infection substrates that include stem loops in the virus genome, and (iii) – in agreement with recent publications (65 68) (Dataset S4). cleavage of substrates in host RNAs. Given the lack of a pheno- Moreover, RNA-Seq revealed greater than 25 transcripts that type in Dicer-deficient cells, and that our Drosha cleavage prod- were highly induced (greater than fivefold) in response to virus ucts are not the obvious substrates for Dicer, we do not suggest infection in the absence of Drosha (Fig. 5D, group 1). In addition that these cleavage products are funneled into the canonical to these up-regulated transcripts, a comparable number of miRNA pathway to directly target viral RNAs. One outstanding mRNAs were down-regulated in virus-infected cells lacking question is how Drosha recognizes its target in the cytoplasm. It is Drosha (group 2) whereas others remained unchanged (a small possible that the nuclease differentiates between host and viral subset of which are depicted as group 3) (Fig. 5D). We also RNA based on adenine/uracil content. Conserved sequence identified more than 25 noncoding RNAs (ncRNAs) of unknown motifs have recently been identified in pri-miRNAs that are be- function, which were significantly up-regulated in virus-infected, lieved to identify hairpins as Drosha targets and potentially sta- Drosha-deficient cells (Fig. S4C). We verified that Drosha was MICROBIOLOGY bilize the reaction (69). It would be interesting to determine lost upon transduction (Fig. S4D) and that Itaga2 was induced by whether these cis-acting elements required for Drosha processing infection and dependent on Drosha whereas Hspa1a was induced are avoided in viral genomes as a mechanism to circumvent and repressed by Drosha using independent quantitative real- Drosha-mediated attenuation. timePCR (Fig. S4 E and F). Taken together, these data demonstrate that Drosha modulates the host transcriptome during virus In summary, this research ascribes a previously unidentified infection. Whether this modulation occurs in the nucleus or cy- function for Drosha in the restriction of RNA virus replication toplasm is unknown. Although future work will be needed to fully and suggests that the enzyme acts independent of its canonical characterize the functional network of Drosha targets in infected role in miRNA biogenesis during virus infection. Taken together, cells, these exciting findings suggest a novel interplay between we speculate that Drosha orchestrates an antiviral response that a miRNA biogenesis factor and the restriction of virus infection. is conserved from arthropods to chordates and is aimed at disrupting both viral and host RNA profiles to attenuate Discussion virus replication. Following the recent evidence for RNAi in mammals and the Materials and Methods discovery that RNA virus infection causes a dramatic change in the subcellular localization of Drosha, we set out to determine Cell Culture, Transfections, and Viruses. For cell culture, transfections, and viruses, see SI Materials and Methods. the range, requirements, and physiological function of this activity. We began our studies by screening a panel of diverse viruses and Drosha in Vitro Cleavage Assay. Drosha in vitro assays were performed as PAMPs, which we found all capable of inducing the relocalization previously described (63). of Drosha to the cytoplasm (Fig. 1). We next investigated the impact of the small RNA biogenesis factors, Drosha and Dicer, on Protein Analysis and Statistics. For protein analysis and statistics, see SI SINV and VSV replication. Although both encoding an RNA Materials and Methods. genome, their mechanisms of replication are very different. Mul- ticycle growth curves in conditional knockout cells identified Dro- Small RNA Northern Blots. Small RNA Northern blots and probe labeling were sha as a restriction factor in virus replication, independent of Dicer performed as previously described (70). Probes used are provided in Dataset and miRNAs (Fig. 2). In an attempt to elucidate the mechanism for S5. All data are representative of at least three independent experiments. Drosha’s antiviral activity, we studied the biology of cDrosha. De- pletion of the nuclear export protein CRM1 resulted in a loss of Small RNA and mRNA Deep Sequencing. For small RNA and mRNA deep se- Drosha-dependent processing of virus-derived pri-miRNAs (Fig. quencing, see SI Materials and Methods. 3). This activity was unaffected by the disruption of Ddx58, Tbk1,or ACKNOWLEDGMENTS. We acknowledge the Mount Sinai Genomics Core Ifnar1 (Fig. S3). Furthermore, we implicated serine 300 and 302 in Facility for deep-sequencing analyses. Rnasenf/f primary lung fibroblasts were the virus-inducible translocation of Drosha and showed that cyto- a kind gift from Dr. Dan Littman (New York University). Monoclonal anti- plasmic localization is required to confer the full antiviral activity of influenza A virus NP antibody, clone IC5-1B7 (produced in vitro, NR-4544) Drosha (Fig. 4). Finally, we investigated whether Drosha was ca- was obtained through Biodefense and Emerging Infections Resources, Na- tional Institute of Allergy and Infectious Diseases, National Institutes of Health pable of generating the small RNAs recently described in fibro- (NIH). Confocal laser-scanning microscopy was performed at the Icahn School blasts (64). In vitro analyses in conjunction with deep sequencing of Medicine at Mount Sinai-Microscopy Shared Resource Facility. This material suggested that Drosha is capable of cleaving viral RNA (Fig. 5). is based upon work supported in part by the US Army Research Laboratory and Small RNA data from both mammalian and insect virus infections the US Army Research Office under Grant W911NF-07-R-0003-4. J.S.S. is supported in part by Public Health Service Institutional Research Training Award showed that loss of Drosha did not impact the small RNA profile of AI07647. B.R.t. is supported in part by the Burroughs Wellcome Fund (BWF). specific viral RNAs, but, rather, loss of Drosha enhanced the S.C. is supported in part by the BWF and NIH Grants R01AI095500 and presence of viral RNA, presumably as a result of increased R01AI074951.

Shapiro et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 1. Hutvágner G, Zamore PD (2002) RNAi: Nature abhors a double-strand. Curr Opin 36. Kelly EJ, Hadac EM, Greiner S, Russell SJ (2008) Engineering microRNA responsiveness Genet Dev 12(2):225–232. to decrease virus pathogenicity. Nat Med 14(11):1278–1283. 2. Sabin LR, et al. (2013) Dicer-2 processes diverse viral RNA species. PLoS ONE 8(2): 37. Barnes D, Kunitomi M, Vignuzzi M, Saksela K, Andino R (2008) Harnessing endoge- e55458. nous miRNAs to control virus tissue tropism as a strategy for developing attenuated 3. Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: Double-stranded RNA directs virus vaccines. Cell Host Microbe 4(3):239–248. the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101(1): 38. Chua MA, Schmid S, Perez JT, Langlois RA, Tenoever BR (2013) Influenza A virus 25–33. utilizes suboptimal splicing to coordinate the timing of infection. Cell Rep 3(1):23–29. 4. Ratcliff FG, MacFarlane SA, Baulcombe DC (1999) Gene silencing without DNA: RNA- 39. Pham AM, Langlois RA, TenOever BR (2012) Replication in cells of hematopoietic mediated cross-protection between viruses. Plant Cell 11(7):1207–1216. origin is necessary for Dengue virus dissemination. PLoS Pathog 8(1):e1002465. 5. Maillard PV, et al. (2013) Antiviral RNA interference in mammalian cells. Science 40. Langlois RA, Varble A, Chua MA, García-Sastre A, tenOever BR (2012) Hematopoietic- 342(6155):235–238. specific targeting of influenza A virus reveals replication requirements for induction 6. Li Y, Lu J, Han Y, Fan X, Ding SW (2013) RNA interference functions as an antiviral of antiviral immune responses. Proc Natl Acad Sci USA 109(30):12117–12122. immunity mechanism in mammals. Science 342(6155):231–234. 41. Langlois RA, et al. (2013) MicroRNA-based strategy to mitigate the risk of gain- 7. Cullen BR, Cherry S, tenOever BR (2013) Is RNA interference a physiologically relevant of-function influenza studies. Nat Biotechnol 31(9):844–847. innate antiviral immune response in mammals? Cell Host Microbe 14(4):374–378. 42. Kawai T, Akira S (2006) Innate immune recognition of viral infection. Nat Immunol 8. Song E, et al. (2003) Sustained small interfering RNA-mediated human immunodefi- 7(2):131–137. ciency virus type 1 inhibition in primary macrophages. J Virol 77(13):7174–7181. 43. Ding SW (2010) RNA-based antiviral immunity. Nat Rev Immunol 10(9):632–644. 9. Novina CD, et al. (2002) siRNA-directed inhibition of HIV-1 infection. Nat Med 8(7): 44. Daher A, et al. (2001) Two dimerization domains in the trans-activation response 681–686. RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of 10. McCaffrey AP, et al. (2003) Inhibition of hepatitis B virus in mice by RNA interference. HIV-1 long terminal repeat expression. J Biol Chem 276(36):33899–33905. Nat Biotechnol 21(6):639–644. 45. Kok KH, et al. (2011) The double-stranded RNA-binding protein PACT functions as 11. Ge Q, et al. (2004) Inhibition of influenza virus production in virus-infected mice by a cellular activator of RIG-I to facilitate innate antiviral response. Cell Host Microbe RNA interference. Proc Natl Acad Sci USA 101(23):8676–8681. 9(4):299–309. 12. Gitlin L, Karelsky S, Andino R (2002) Short interfering RNA confers intracellular an- 46. Ohman M (2007) A-to-I editing challenger or ally to the microRNA process. Biochimie tiviral immunity in human cells. Nature 418(6896):430–434. 89(10):1171–1176. 13. Ge Q, et al. (2003) RNA interference of influenza virus production by directly tar- 47. Ota H, et al. (2013) ADAR1 forms a complex with Dicer to promote microRNA pro- geting mRNA for degradation and indirectly inhibiting all viral RNA transcription. cessing and RNA-induced gene silencing. Cell 153(3):575–589. Proc Natl Acad Sci USA 100(5):2718–2723. 48. Cullen BR (2013) MicroRNAs as mediators of viral evasion of the immune system. Nat 14. Bitko V, Barik S (2001) Phenotypic silencing of cytoplasmic genes using sequence- Immunol 14(3):205–210. specific double-stranded short interfering RNA and its application in the reverse ge- 49. Pfeffer S, et al. (2004) Identification of virus-encoded microRNAs. Science 304(5671): netics of wild type negative-strand RNA viruses. BMC Microbiol 1:34. 734–736. 15. Coburn GA, Cullen BR (2002) Potent and specific inhibition of human immunodefi- 50. Kincaid RP, Sullivan CS (2012) Virus-encoded microRNAs: An overview and a look to ciency virus type 1 replication by RNA interference. J Virol 76(18):9225–9231. the future. PLoS Pathog 8(12):e1003018. 16. Parameswaran P, et al. (2010) Six RNA viruses and forty-one hosts: Viral small RNAs 51. Kincaid RP, Burke JM, Sullivan CS (2012) RNA virus microRNA that mimics a B-cell and modulation of small RNA repertoires in vertebrate and invertebrate systems. oncomiR. Proc Natl Acad Sci USA 109(8):3077–3082. PLoS Pathog 6(2):e1000764. 52. Flemr M, et al. (2013) A retrotransposon-driven dicer isoform directs endogenous 17. Girardi E, Chane-Woon-Ming B, Messmer M, Kaukinen P, Pfeffer S (2013) Identifica- small interfering RNA production in mouse oocytes. Cell 155(4):807–816. tion of RNase L-dependent, 3′-end-modified, viral small RNAs in Sindbis virus-infected 53. Shapiro JS, Varble A, Pham AM, Tenoever BR (2010) Noncanonical cytoplasmic pro- mammalian cells. MBio 4(6):e00698–e13. cessing of viral microRNAs. RNA 16(11):2068–2074. 18. Bowie AG, Unterholzner L (2008) Viral evasion and subversion of pattern-recognition 54. Rouha H, Thurner C, Mandl CW (2010) Functional microRNA generated from a cyto- receptor signalling. Nat Rev Immunol 8(12):911–922. plasmic RNA virus. Nucleic Acids Res 38(22):8328–8337. 19. Sharma S, et al. (2003) Triggering the interferon antiviral response through an IKK- 55. Langlois RA, Shapiro JS, Pham AM, tenOever BR (2012) In vivo delivery of cytoplasmic related pathway. Science 300(5622):1148–1151. RNA virus-derived miRNAs. Mol Ther 20(2):367–375. 20. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary 56. Varble A, et al. (2013) An in vivo RNAi screening approach to identify host determi- microRNAs by the Microprocessor complex. Nature 432(7014):231–235. nants of virus replication. Cell Host Microbe 14(3):346–356. 21. Gregory RI, et al. (2004) The Microprocessor complex mediates the genesis of mi- 57. Backes S, et al. (2012) Degradation of host microRNAs by poxvirus poly(A) polymerase croRNAs. Nature 432(7014):235–240. reveals terminal RNA methylation as a protective antiviral mechanism. Cell Host Mi- 22. Han J, et al. (2006) Molecular basis for the recognition of primary microRNAs by the crobe 12(2):200–210. Drosha-DGCR8 complex. Cell 125(5):887–901. 58. Shapiro JS, Langlois RA, Pham AM, Tenoever BR (2012) Evidence for a cytoplasmic 23. Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell microprocessor of pri-miRNAs. RNA 18(7):1338–1346. Biol 10(2):126–139. 59. García-Sastre A, et al. (1998) Influenza A virus lacking the NS1 gene replicates in in- 24. Park JE, et al. (2011) Dicer recognizes the 5′ end of RNA for efficient and accurate terferon-deficient systems. Virology 252(2):324–330. processing. Nature 475(7355):201–205. 60. Rose PP, et al. (2011) Natural resistance-associated macrophage protein is a cellular 25. Hutvágner G, et al. (2001) A cellular function for the RNA-interference enzyme Dicer receptor for sindbis virus in both insect and mammalian hosts. Cell Host Microbe in the maturation of the let-7 small temporal RNA. Science 293(5531):834–838. 10(2):97–104. 26. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribo- 61. Kuhn RJ, ed (2007) Togaviridae: The Viruses and Their Replication (Lippincott Williams nuclease in the initiation step of RNA interference. Nature 409(6818):363–366. & Wilkins, Philadelphia), 5th Ed, pp 1002–1022. 27. Ebert MS, Sharp PA (2012) Roles for microRNAs in conferring robustness to biological 62. Tang X, Zhang Y, Tucker L, Ramratnam B (2010) Phosphorylation of the RNase III processes. Cell 149(3):515–524. enzyme Drosha at Serine300 or Serine302 is required for its nuclear localization. 28. Czech B, Hannon GJ (2011) Small RNA sorting: Matchmaking for Argonautes. Nat Rev Nucleic Acids Res 38(19):6610–6619. Genet 12(1):19–31. 63. Lee Y, et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 29. Zekri L, Huntzinger E, Heimstädt S, Izaurralde E (2009) The silencing domain of 425(6956):415–419. GW182 interacts with PABPC1 to promote translational repression and degradation 64. Donaszi-Ivanov A, Mohorianu I, Dalmay T, Powell PP. Small RNA analysis in Sindbis of microRNA targets and is required for target release. Mol Cell Biol 29(23): virus infected human HEK293 cells. PLoS ONE 8(12):e84070. 6220–6231. 65. Han J, et al. (2009) Posttranscriptional crossregulation between Drosha and DGCR8. 30. Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian microRNAs pre- Cell 136(1):75–84. dominantly act to decrease target mRNA levels. Nature 466(7308):835–840. 66. Chong MM, et al. (2010) Canonical and alternate functions of the microRNA bio- 31. Djuranovic S, Nahvi A, Green R (2012) miRNA-mediated gene silencing by trans- genesis machinery. Genes Dev 24(17):1951–1960. lational repression followed by mRNA deadenylation and decay. Science 336(6078): 67. Karginov FV, et al. (2010) Diverse endonucleolytic cleavage sites in the mammalian 237–240. transcriptome depend upon microRNAs, Drosha, and additional nucleases. Mol Cell 32. Bazzini AA, Lee MT, Giraldez AJ (2012) Ribosome profiling shows that miR-430 reduces 38(6):781–788. translation before causing mRNA decay in zebrafish. Science 336(6078):233–237. 68. Lin YT, Sullivan CS (2011) Expanding the role of Drosha to the regulation of viral gene 33. tenOever BR (2013) RNA viruses and the host microRNA machinery. Nat Rev Microbiol expression. Proc Natl Acad Sci USA 108(27):11229–11234. 11(3):169–180. 69. Auyeung VC, Ulitsky I, McGeary SE, Bartel DP (2013) Beyond secondary structure: 34. Edge RE, et al. (2008) A let-7 MicroRNA-sensitive vesicular stomatitis virus demon- Primary-sequence determinants license pri-miRNA hairpins for processing. Cell 152(4): strates tumor-specific replication. Mol Ther 16(8):1437–1443. 844–858. 35. Perez JT, et al. (2009) MicroRNA-mediated species-specific attenuation of influenza A 70. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection virus. Nat Biotechnol 27(6):572–576. of small RNA. Nat Protoc 3(6):1077–1084.

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