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 a Department 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 restricted to plants, nematodes, and arthropods. In an effort to determine whether a physiological interplay exists between the host small RNA machinery and the cellular response to virus infection in mammals, we evaluated antiviral activity in the presence and absence of Dicer or Drosha, the RNase III nucleases responsible for generating small RNAs. Although loss of Dicer did not compromise the cellular response to virus infection, Drosha deletion resulted in a significant increase in virus levels. Here, we demonstrate that diverse RNA viruses trigger exportin 1 (XPO1/CRM1)-dependent Drosha translocation into the cytoplasm in a manner independent of de novo protein synthesis or the canonical type I IFN system. Additionally, increased virus infection in the absence of Drosha was not due to a loss of viral small RNAs but, instead, correlated with cleavage of viral genomic RNA and modulation of the host transcriptome. Taken together, we propose that Drosha represents a unique and conserved arm of the cellular defenses used to combat virus infection. RNAi
| microRNA | miRNA | Rnasen | innate immunity
I
n plants, nematodes, and arthropods, a major response to virus infection is Dicer-dependent generation of virus-derived small interfering RNAs (vsiRNAs) (1, 2). vsiRNAs associate with the RNA-induced silencing complex (RISC) and mediate cleavage of homologous viral RNA, attenuating virus replication in a process termed antiviral RNA interference (RNAi) (3, 4). Although many components of antiviral RNAi are conserved in chordates, the small RNA-mediated response to virus infection has largely been replaced with the protein-based type I IFN (IFN-I) response although evidence for mammalian RNAi has recently been reported in some cell types against particular viruses (5–7). Indeed, ectopic expression of siRNAs directed against viral genomes in diverse cell types potently inhibits virus replication of a wide range of viruses (8–15). However, vsiRNAs have been difficult to detect in IFN-I–sensitive cells (2, 16, 17). These data suggest that, whereas chordates may not produce robust levels of vsiRNAs, they are capable of harnessing the small RNA machinery in an antiviral capacity when presented with the proper substrate. These same data also suggest that mammalian RNA viruses have not incurred any clear selective pressure to inhibit small RNA-mediated signaling, in contrast to the IFN-I induction pathway where virus antagonism is common (18). In mammals, RNA virus infection is recognized in response to replication, as this process generates a diverse array of pathogen associated molecular patterns (PAMPs). PAMPs include double stranded RNA (dsRNA), RNA with an exposed 5′ triphosphate, or RNA lacking a 2′ O-methyl–containing cap (18). In the vast majority of cells, PAMPs are detected by one of the two PAMP recognition receptors (PRRs): RIG-I (Encoded by the Ddx58 gene) and MDA5 (18). PRR detection culminates in a signal transduction event that includes activation of the IFN regulatory factors (IRFs) by tank-binding kinase 1 (TBK1) (19). Kinase activation results in assembly of a multisubunit enhancer that promotes transcription of the IFN beta gene, a member of the IFN-I family. IFN-I production subsequently results in the up-regulation
7108–7113 | PNAS | May 13, 2014 | vol. 111 | no. 19
of hundreds of IFN-stimulated genes (ISGs) through a ubiquitous IFN-I receptor (encoded by the Ifnar1 gene) (18). Despite the lack of robust vsiRNA production, chordates have retained genome-encoded microRNAs (miRNAs). These noncoding RNAs are transcribed by RNA polymerase II and processed in a stepwise fashion by two RNase III enzymes: first, Drosha in the nucleus; and second, Dicer in the cytoplasm (20–26). Similar to vsiRNAs, miRNAs are also capable of exerting RNAi although they more commonly act to fine-tune host gene expression through translational repression and/or mRNA deadenylation and are thought to contribute to cellular fitness (27–32). Given the modest repression of miRNAs on their targets, a property that results from imperfect binding complementarity, they are unlikely to serve as direct inhibitors of viral transcripts (33). However, viruses can be engineered to encode perfect complementary target sites for endogenous miRNAs as an effective mechanism to attenuate virus replication (34–41). Despite the apparent evolutionary loss of vsiRNAs as an antiviral defense in chordates, there are many overlaps between the RNAi and IFN-I pathways, most notable being that both IFN-I and RNAi can be triggered by the presence of dsRNA (42, 43). Furthermore, a number of proteins involved in miRNA production have also been implicated in the IFN-I response. For example, the dsRNA-binding proteins TRBP and PACT, which aid in precursor-miRNA dicing, RISC maturation, and target silencing, have also been reported to inhibit and activate effectors of the IFN-I pathway, respectively (44, 45). In addition, both the ubiquitous and IFN-I–inducible isoform of ADAR1 can function to alter miRNA expression (46) and associate with Dicer to enhance enzyme activity (47). Conversely, many viruses interact with Drosha and Dicer for the production of viral miRNAs or to regulate the levels of viral transcripts (48–51). The range in interplay between virus and the mammalian miRNA pathway demonstrates the capacity for cross-talk between these two systems, but the physiological relevance of this cross-talk remains Significance Virus infections must be combated at a cellular level. The strategies used to inhibit virus differ dramatically when comparing plants and insects to mammals. Here, we identify an evolutionary conserved antiviral response that is independent of these known defenses. We demonstrate that an RNA nuclease called Drosha is repurposed during virus infection to cleave viral RNA and modulate the cellular environment as a means of inhibiting virus replication. Author contributions: J.S.S., S.S., L.R.S., and B.R.t. designed research; J.S.S., S.S., L.C.A., 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., S.C., and B.R.t. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. B.R.C. is a guest editor invited by the Editorial Board. 1
J.S.S. and S.S. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1319635111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1319635111
Results Drosha Translocation Is a General Response to RNA Virus Infections.
Recent evidence has demonstrated the capacity to engineer cytoplasmic viruses to produce miRNAs (53–57). Subsequently, we found that cytoplasmic miRNA synthesis was dependent on a Drosha translocation event to process the miRNA from Sindbis virus (SINV) (58). Given the recent findings relating to the ability of the miRNA machinery to naturally exert an antiviral response in mammalian fibroblasts (6), we sought to investigate whether the SINV-induced translocation of Drosha into the cytoplasm represents a broad antiviral response. Therefore, we investigated Drosha localization in response to infection with a positive sense virus (SINV), a negative sense virus [vesicular stomatitis virus (VSV)], and a nuclear, segmented RNA virus [mutated influenza A virus (mIAV)], which lacks its main antagonist of the antiviral response [nonstructural protein (NS1), described in ref. 59], and in response to treatment with the canonical viral PAMP, dsRNA (Fig. 1A). Interestingly, we found that, despite exclusive expression of Drosha in the nucleus in mock-treated cells, there was robust translocation to the cytoplasm in response to SINV, VSV, mIAV, or dsRNA (Fig. 1A). Furthermore, cytoplasmic Drosha (cDrosha) was evident during the early hours of infection and dsRNA treatment (Fig. 1A). These data suggest that detection of a broad range of viral PAMPs results in the accumulation of Drosha in the cytoplasm. Drosha-Dependent Cytoplasmic miRNA Processing Is Conserved in Arthropods. Given the generality of virus-induced Drosha trans-
location, we next assessed whether insects also display cDrosha activity by assaying miRNA production from a Drosha-dependent, cytoplasmic RNA transcript. Drosophila melanogaster cells are permissive hosts of many alphaviruses and, as in mammalian cells, support a cytoplasmic SINV replication cycle (60). As such, Drosophila cells (DL1) were infected with a recombinant SINV encoding primary (pri)-miR-124 (SINV124) (53), and miR-124 synthesis from the cytoplasmic transcript was monitored. Similar to mammalian infections, SINV124 resulted in miRNA
Fig. 1. Broad accumulation of cDrosha in virus-infected cells. (A) Immunohistochemistry of murine fibroblasts infected with SINV (MOI = 3), VSV (MOI = 1), or mIAV lacking the main viral IFN antagonist nonstructural protein NS1 (MOI = 5), or transfected with poly(I:C) for 6 h and stained for 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 for the indicated times. (C) Small RNA Northern blot of DL1 cells treated with dsRNA against β-galactosidase (bgal) or Drosha and subsequently infected with SINV124.
Shapiro et al.
biogenesis throughout the course of the Drosophila cell infection (Fig. 1B). Next, to determine whether this activity was also Drosha-dependent, we depleted cells of this nuclease and assessed cytoplasmic (c)-pri-miR-124 processing (Fig. 1C and Fig. S1). Drosha depletion abrogated the ability of SINV124 to produce a mature miRNA and resulted in an enhancement in the level of unprocessed virus-derived pri-miR-124 and viral RNA (Fig. 1C and Fig. S1). Taken together, these data implicate a possible role for Drosha in the cleavage of the cytoplasmic virus-derived RNA transcripts in Drosophila and suggest increased virus replication in the absence of the nuclease. Sindbis Virus Is Susceptible to an RNAi-Mediated Antiviral Response.
Given the broad accumulation of cDrosha in response to a panel of RNA viruses, we hypothesized that Drosha may play a role in an RNAi-like response perhaps related to the recent findings in mammalian cells (5–7). We hypothesized that, if Drosha had a role in antiviral RNAi, then both Drosha and Dicer would have antiviral properties where products from Drosha would be fed to Dicer, which would then produce substrates for RISC that would silence viral RNAs. To determine whether these two RNase III nucleases, Drosha and Dicer, contribute to the cellular response to virus infection, we measured the impact of virus replication in the presence or absence of each nuclease. We chose to further study SINV as it is a virus model that has previously been shown to be capable of generating miRNAs, suggesting that it does not disrupt the host machinery responsible for small RNA biogenesis (53). However, to first ensure that the virus lacked a suppressor of RNA silencing (SRS), we assessed whether small RNAs could be harnessed to inhibit SINV replication by engineering the virus with a scrambled RNA (scbl) in its 3′ UTR or two or four miR-124 target sites in the same location (2 × 124T, or 4 × 124T, respectively). Because miR-124 is restricted to neurons, infection of SINV scbl, 2 × 124T, or 4 × 124T in fibroblasts resulted in equal levels of SINV replication as measured by capsid protein synthesis (Fig. 2A). In contrast, exogenous expression of miR-124 resulted in a complete loss of capsid expression in 2 × 124T or 4 × 124T virus while having no impact on SINV scbl or on host protein disulfide isomerase (PDI) (Fig. 2A). Taken together, these results suggest that SINV is capable of being targeted by RNAi during infection. Loss of Drosha Results in an Increase in RNA Virus Replication. Given the lack of a SINV SRS, we next used conditional knockout fibroblasts for Drosha or Dicer (Rnasenf/f and Dicer1f/f, respectively) and disrupted each gene using replication-incompetent Adenobased 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 capsid protein also accumulated to higher quantities in Droshadepleted cells, consistent with elevated levels of virus replication (Fig. 2D). In contrast, depletion of Dicer did not result in a significant alteration in the SINV titers over the course of infection compared with control cells (Fig. 2E). Western blot also revealed unaltered virus levels between Dicer-deficient and control cells (Fig. 2F). Furthermore, VSV also displayed enhanced replication in the absence of Drosha but not Dicer (Fig. S2 A–D). These data demonstrate that Drosha restricts PNAS | May 13, 2014 | vol. 111 | no. 19 | 7109
MICROBIOLOGY
poorly understood. Supporting data for direct RNAi against viral RNAs in mammalian cells includes evidence for RNase III-like activity in the restriction of retrotransposons and two RNA virus infection models (5, 6, 52). Given these findings and associations, we sought to determine whether Dicer or Drosha, the only mammalian RNase III nucleases, contributed to the mammalian response to virus infection in somatic cells, which are the major targets of viral infection.
Fig. 2. Drosha restricts virus replication. (A) 293T cells transfected with empty or miR-124 producing vector for 36 h and subsequently infected with SINV expressing a scrambled sequence (scbl) or two or four miR-124 target 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 Northern blot of ectopically expressed miR-124. (B) Small RNA Northern blot of Rnasenf/f or Dicer1f/f fibroblasts treated with replication-incompetent Adeno-based vectors (AdV) expressing GFP or a GFP-Cre fusion protein (Cre) for 5 d and probed for miR-93 (Upper) or U6 (Lower). (C) Plaque assay of Rnasenf/f fibroblasts treated with AdV-GFP or AdV-Cre and subsequently infected with recombinant SINV (MOI = 0.1). (D) Western blot for same conditions as in C. (E) Plaque assay of Dicer1f/f fibroblasts treated with AdVGFP or AdV-Cre and subsequently infected with recombinant SINV (MOI = 0.1). (F) Western blot for same conditions as in E. Data in C and E are represented as the mean ± SEM for n = 3. *Significant P value of
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 restricted to plants, nematodes, and arthropods. In an effort to determine whether a physiological interplay exists between the host small RNA machinery and the cellular response to virus infection in mammals, we evaluated antiviral activity in the presence and absence of Dicer or Drosha, the RNase III nucleases responsible for generating small RNAs. Although loss of Dicer did not compromise the cellular response to virus infection, Drosha deletion resulted in a significant increase in virus levels. Here, we demonstrate that diverse RNA viruses trigger exportin 1 (XPO1/CRM1)-dependent Drosha translocation into the cytoplasm in a manner independent of de novo protein synthesis or the canonical type I IFN system. Additionally, increased virus infection in the absence of Drosha was not due to a loss of viral small RNAs but, instead, correlated with cleavage of viral genomic RNA and modulation of the host transcriptome. Taken together, we propose that Drosha represents a unique and conserved arm of the cellular defenses used to combat virus infection. RNAi
| microRNA | miRNA | Rnasen | innate immunity
I
n plants, nematodes, and arthropods, a major response to virus infection is Dicer-dependent generation of virus-derived small interfering RNAs (vsiRNAs) (1, 2). vsiRNAs associate with the RNA-induced silencing complex (RISC) and mediate cleavage of homologous viral RNA, attenuating virus replication in a process termed antiviral RNA interference (RNAi) (3, 4). Although many components of antiviral RNAi are conserved in chordates, the small RNA-mediated response to virus infection has largely been replaced with the protein-based type I IFN (IFN-I) response although evidence for mammalian RNAi has recently been reported in some cell types against particular viruses (5–7). Indeed, ectopic expression of siRNAs directed against viral genomes in diverse cell types potently inhibits virus replication of a wide range of viruses (8–15). However, vsiRNAs have been difficult to detect in IFN-I–sensitive cells (2, 16, 17). These data suggest that, whereas chordates may not produce robust levels of vsiRNAs, they are capable of harnessing the small RNA machinery in an antiviral capacity when presented with the proper substrate. These same data also suggest that mammalian RNA viruses have not incurred any clear selective pressure to inhibit small RNA-mediated signaling, in contrast to the IFN-I induction pathway where virus antagonism is common (18). In mammals, RNA virus infection is recognized in response to replication, as this process generates a diverse array of pathogen associated molecular patterns (PAMPs). PAMPs include double stranded RNA (dsRNA), RNA with an exposed 5′ triphosphate, or RNA lacking a 2′ O-methyl–containing cap (18). In the vast majority of cells, PAMPs are detected by one of the two PAMP recognition receptors (PRRs): RIG-I (Encoded by the Ddx58 gene) and MDA5 (18). PRR detection culminates in a signal transduction event that includes activation of the IFN regulatory factors (IRFs) by tank-binding kinase 1 (TBK1) (19). Kinase activation results in assembly of a multisubunit enhancer that promotes transcription of the IFN beta gene, a member of the IFN-I family. IFN-I production subsequently results in the up-regulation
7108–7113 | PNAS | May 13, 2014 | vol. 111 | no. 19
of hundreds of IFN-stimulated genes (ISGs) through a ubiquitous IFN-I receptor (encoded by the Ifnar1 gene) (18). Despite the lack of robust vsiRNA production, chordates have retained genome-encoded microRNAs (miRNAs). These noncoding RNAs are transcribed by RNA polymerase II and processed in a stepwise fashion by two RNase III enzymes: first, Drosha in the nucleus; and second, Dicer in the cytoplasm (20–26). Similar to vsiRNAs, miRNAs are also capable of exerting RNAi although they more commonly act to fine-tune host gene expression through translational repression and/or mRNA deadenylation and are thought to contribute to cellular fitness (27–32). Given the modest repression of miRNAs on their targets, a property that results from imperfect binding complementarity, they are unlikely to serve as direct inhibitors of viral transcripts (33). However, viruses can be engineered to encode perfect complementary target sites for endogenous miRNAs as an effective mechanism to attenuate virus replication (34–41). Despite the apparent evolutionary loss of vsiRNAs as an antiviral defense in chordates, there are many overlaps between the RNAi and IFN-I pathways, most notable being that both IFN-I and RNAi can be triggered by the presence of dsRNA (42, 43). Furthermore, a number of proteins involved in miRNA production have also been implicated in the IFN-I response. For example, the dsRNA-binding proteins TRBP and PACT, which aid in precursor-miRNA dicing, RISC maturation, and target silencing, have also been reported to inhibit and activate effectors of the IFN-I pathway, respectively (44, 45). In addition, both the ubiquitous and IFN-I–inducible isoform of ADAR1 can function to alter miRNA expression (46) and associate with Dicer to enhance enzyme activity (47). Conversely, many viruses interact with Drosha and Dicer for the production of viral miRNAs or to regulate the levels of viral transcripts (48–51). The range in interplay between virus and the mammalian miRNA pathway demonstrates the capacity for cross-talk between these two systems, but the physiological relevance of this cross-talk remains Significance Virus infections must be combated at a cellular level. The strategies used to inhibit virus differ dramatically when comparing plants and insects to mammals. Here, we identify an evolutionary conserved antiviral response that is independent of these known defenses. We demonstrate that an RNA nuclease called Drosha is repurposed during virus infection to cleave viral RNA and modulate the cellular environment as a means of inhibiting virus replication. Author contributions: J.S.S., S.S., L.R.S., and B.R.t. designed research; J.S.S., S.S., L.C.A., 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., S.C., and B.R.t. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. B.R.C. is a guest editor invited by the Editorial Board. 1
J.S.S. and S.S. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1319635111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1319635111
Results Drosha Translocation Is a General Response to RNA Virus Infections.
Recent evidence has demonstrated the capacity to engineer cytoplasmic viruses to produce miRNAs (53–57). Subsequently, we found that cytoplasmic miRNA synthesis was dependent on a Drosha translocation event to process the miRNA from Sindbis virus (SINV) (58). Given the recent findings relating to the ability of the miRNA machinery to naturally exert an antiviral response in mammalian fibroblasts (6), we sought to investigate whether the SINV-induced translocation of Drosha into the cytoplasm represents a broad antiviral response. Therefore, we investigated Drosha localization in response to infection with a positive sense virus (SINV), a negative sense virus [vesicular stomatitis virus (VSV)], and a nuclear, segmented RNA virus [mutated influenza A virus (mIAV)], which lacks its main antagonist of the antiviral response [nonstructural protein (NS1), described in ref. 59], and in response to treatment with the canonical viral PAMP, dsRNA (Fig. 1A). Interestingly, we found that, despite exclusive expression of Drosha in the nucleus in mock-treated cells, there was robust translocation to the cytoplasm in response to SINV, VSV, mIAV, or dsRNA (Fig. 1A). Furthermore, cytoplasmic Drosha (cDrosha) was evident during the early hours of infection and dsRNA treatment (Fig. 1A). These data suggest that detection of a broad range of viral PAMPs results in the accumulation of Drosha in the cytoplasm. Drosha-Dependent Cytoplasmic miRNA Processing Is Conserved in Arthropods. Given the generality of virus-induced Drosha trans-
location, we next assessed whether insects also display cDrosha activity by assaying miRNA production from a Drosha-dependent, cytoplasmic RNA transcript. Drosophila melanogaster cells are permissive hosts of many alphaviruses and, as in mammalian cells, support a cytoplasmic SINV replication cycle (60). As such, Drosophila cells (DL1) were infected with a recombinant SINV encoding primary (pri)-miR-124 (SINV124) (53), and miR-124 synthesis from the cytoplasmic transcript was monitored. Similar to mammalian infections, SINV124 resulted in miRNA
Fig. 1. Broad accumulation of cDrosha in virus-infected cells. (A) Immunohistochemistry of murine fibroblasts infected with SINV (MOI = 3), VSV (MOI = 1), or mIAV lacking the main viral IFN antagonist nonstructural protein NS1 (MOI = 5), or transfected with poly(I:C) for 6 h and stained for 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 for the indicated times. (C) Small RNA Northern blot of DL1 cells treated with dsRNA against β-galactosidase (bgal) or Drosha and subsequently infected with SINV124.
Shapiro et al.
biogenesis throughout the course of the Drosophila cell infection (Fig. 1B). Next, to determine whether this activity was also Drosha-dependent, we depleted cells of this nuclease and assessed cytoplasmic (c)-pri-miR-124 processing (Fig. 1C and Fig. S1). Drosha depletion abrogated the ability of SINV124 to produce a mature miRNA and resulted in an enhancement in the level of unprocessed virus-derived pri-miR-124 and viral RNA (Fig. 1C and Fig. S1). Taken together, these data implicate a possible role for Drosha in the cleavage of the cytoplasmic virus-derived RNA transcripts in Drosophila and suggest increased virus replication in the absence of the nuclease. Sindbis Virus Is Susceptible to an RNAi-Mediated Antiviral Response.
Given the broad accumulation of cDrosha in response to a panel of RNA viruses, we hypothesized that Drosha may play a role in an RNAi-like response perhaps related to the recent findings in mammalian cells (5–7). We hypothesized that, if Drosha had a role in antiviral RNAi, then both Drosha and Dicer would have antiviral properties where products from Drosha would be fed to Dicer, which would then produce substrates for RISC that would silence viral RNAs. To determine whether these two RNase III nucleases, Drosha and Dicer, contribute to the cellular response to virus infection, we measured the impact of virus replication in the presence or absence of each nuclease. We chose to further study SINV as it is a virus model that has previously been shown to be capable of generating miRNAs, suggesting that it does not disrupt the host machinery responsible for small RNA biogenesis (53). However, to first ensure that the virus lacked a suppressor of RNA silencing (SRS), we assessed whether small RNAs could be harnessed to inhibit SINV replication by engineering the virus with a scrambled RNA (scbl) in its 3′ UTR or two or four miR-124 target sites in the same location (2 × 124T, or 4 × 124T, respectively). Because miR-124 is restricted to neurons, infection of SINV scbl, 2 × 124T, or 4 × 124T in fibroblasts resulted in equal levels of SINV replication as measured by capsid protein synthesis (Fig. 2A). In contrast, exogenous expression of miR-124 resulted in a complete loss of capsid expression in 2 × 124T or 4 × 124T virus while having no impact on SINV scbl or on host protein disulfide isomerase (PDI) (Fig. 2A). Taken together, these results suggest that SINV is capable of being targeted by RNAi during infection. Loss of Drosha Results in an Increase in RNA Virus Replication. Given the lack of a SINV SRS, we next used conditional knockout fibroblasts for Drosha or Dicer (Rnasenf/f and Dicer1f/f, respectively) and disrupted each gene using replication-incompetent Adenobased 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 capsid protein also accumulated to higher quantities in Droshadepleted cells, consistent with elevated levels of virus replication (Fig. 2D). In contrast, depletion of Dicer did not result in a significant alteration in the SINV titers over the course of infection compared with control cells (Fig. 2E). Western blot also revealed unaltered virus levels between Dicer-deficient and control cells (Fig. 2F). Furthermore, VSV also displayed enhanced replication in the absence of Drosha but not Dicer (Fig. S2 A–D). These data demonstrate that Drosha restricts PNAS | May 13, 2014 | vol. 111 | no. 19 | 7109
MICROBIOLOGY
poorly understood. Supporting data for direct RNAi against viral RNAs in mammalian cells includes evidence for RNase III-like activity in the restriction of retrotransposons and two RNA virus infection models (5, 6, 52). Given these findings and associations, we sought to determine whether Dicer or Drosha, the only mammalian RNase III nucleases, contributed to the mammalian response to virus infection in somatic cells, which are the major targets of viral infection.
Fig. 2. Drosha restricts virus replication. (A) 293T cells transfected with empty or miR-124 producing vector for 36 h and subsequently infected with SINV expressing a scrambled sequence (scbl) or two or four miR-124 target 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 Northern blot of ectopically expressed miR-124. (B) Small RNA Northern blot of Rnasenf/f or Dicer1f/f fibroblasts treated with replication-incompetent Adeno-based vectors (AdV) expressing GFP or a GFP-Cre fusion protein (Cre) for 5 d and probed for miR-93 (Upper) or U6 (Lower). (C) Plaque assay of Rnasenf/f fibroblasts treated with AdV-GFP or AdV-Cre and subsequently infected with recombinant SINV (MOI = 0.1). (D) Western blot for same conditions as in C. (E) Plaque assay of Dicer1f/f fibroblasts treated with AdVGFP or AdV-Cre and subsequently infected with recombinant SINV (MOI = 0.1). (F) Western blot for same conditions as in E. Data in C and E are represented as the mean ± SEM for n = 3. *Significant P value of
