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Mol Ther. Feb 2012; 20(2): 367–375.
Published online Nov 15, 2011. doi:  10.1038/mt.2011.244
PMCID: PMC3277236

In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs

Abstract

The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control used by the cell to maintain optimal protein levels. This process has represented an attractive strategy for therapeutics that is currently limited by in vivo delivery constraints. Here, we describe the generation of a single-stranded, cytoplasmic virus of negative polarity capable of producing functional miRNAs. Cytoplasmic RNA virus-derived miRNAs accumulated to high levels in vitro, generated significant amounts of miRNA star strand, associated with the RNA-induced silencing complex (RISC), and conferred post transcriptional gene silencing in a sequence-specific manner. Furthermore, we demonstrate that these vectors could deliver miRNAs to a wide range of tissues, and sustain prolonged expression capable of achieving measurable knockdown of physiological targets in vivo. Taken together, these results validate noncanonical processing of cytoplasmic-derived miRNAs and provide a novel platform for small RNA delivery.

Introduction

MicroRNAs (miRNA) are small, ~20 nucleotide noncoding, regulatory RNAs important in cellular processes ranging from development to apoptosis.1 miRNAs are transcribed by RNA polymerase II, to generate the primary miRNA (pri-miRNA).1 The nuclear microprocessor, comprised of the ribonuclease (RNase) III, Drosha, and its double-stranded RNA-binding partner, DGCR8, recognizes the pri-miRNA secondary structure, cleaving at the base of the hairpin, yielding a 60–70 nucleotide precursor miRNA (pre-miRNA) with a 2nt 3′ overhang.2,3 Once exported into the cytoplasm, the pre-miRNA is processed by a second RNase III enzyme, Dicer, in conjunction with its double-stranded RNA-binding partner, TRBP, resulting in the generation of a mature miRNA:miRNA star duplex each end having a 2nt 3′ overhang.4–6 Subsequent to Dicer cleavage, the duplex associates with one of the four argonaute proteins (Ago 1–4), a major component of the RNA-induced silencing complex (RISC). Upon RISC maturation the duplex undergoes strand selection. Although not completely understood, this process is based, in part, on 5′ thermodynamic stability of each end of the RNA duplex, with lower mean free energy showing a bias for RISC loading.7,8 The star strand, defined by the strand predominantly expelled from RISC, is degraded.8 The mature miRNA strand loaded into RISC, mediates post-transcriptional silencing (PTS) through partial complementary base-pairing between the miRNA and target mRNA, resulting in RNA destabilization and/or translational repression.1

In addition to the canonical biogenesis pathway described above, we, and others, have recently demonstrated that pri-miRNA, driven from cytoplasmic RNA viruses of positive polarity, can yield mature miRNAs, termed virtrons, exclusively within the cytoplasm.9,10 However, despite extensive deep sequencing efforts from RNA virus infections,11,12 bona fide, pathogen-derived miRNAs remain exclusive to large DNA viruses, suggesting that exploitation of this host function may not be advantageous to cytoplasmic RNA viruses. While there are ongoing efforts questioning the existence of endogenous virtrons, the mechanism by which nuclear-independent miRNAs are generated also remains elusive. One possible mechanism would include a novel cytoplasmic small RNA biogenesis pathway for the processing of pri- to mature miRNAs. Alternatively, it remains a formal possibility that the two characterized examples of virtron production are artifacts of transformed cells, whose rapid division may permit virus mRNA transcripts access to the nuclear microprocessor.13

Given the fundamental importance of discerning whether cytoplasmic generation of miRNAs represents a bona fide novel biological process, and the obvious therapeutic implications such vectors would provide, we sought to engineer a cytoplasmic virus of negative polarity to produce miRNAs and determine whether in vivo delivery could be accomplished. In this report, we demonstrate that cytoplasmic virus vectors, of both positive and negative polarity, are capable of producing miRNAs in vivo. Furthermore, in vivo expression can be sustained beyond five days post infection, despite viral clearance, and can confer PTS in a miRNA-specific manner. Taken together, the work presented herein validates the generation of cytoplasmic miRNAs as an endogenous cellular process that can be exploited as a novel strategy for vector-based small RNA delivery.

Results

Cytoplasmic-mediated synthesis of viral miRNAs

Previous results from our laboratory have demonstrated that influenza A virus (IAV), a nuclear negative-stranded RNA virus, and Sindbis virus (SV), a cytoplasmic positive sense RNA virus, can be engineered to produce a mature miRNA.10,14 While IAV-mediated production of miRNAs was not surprising, given that this virus replicates in the nucleus, comparable production from cytoplasmic SV-derived miRNAs was intriguing as this vector would not access the nuclear microprocessor.10,14 Therefore, we sought to determine whether a cytoplasmic virus of negative polarity could also be generated to produce a miRNA. To this end, we engineered vesicular stomatitis virus (VSV) to encode the neuron-specific miR-12415 as an independent virus transcript (VSV124). The mmu-miR-124-2 locus was inserted between the glycoprotein (G) and large polymerase (L) genes, and was flanked by the necessary 5′ cap and 3′ poly(A) signals needed for polymerase recognition16 (Figure 1a). Insertion of the hairpin did not impede the rescue of recombinant virus and, following infection, led to the production of virus-derived miR-124, producing a small RNA, similar in size to plasmid-derived miR-124 (Figure 1b). Infection with VSV124 did not alter miR-93 expression, a ubiquitous miRNA used as a loading control. Additionally, VSV infection did not induce the production of endogenous miR-124, as VSV containing a noncoding scrambled RNA (herein referred to as VSVscbl) failed to induce any detectable miR-124 despite reaching comparable levels of replication (Figure 1c).

Figure 1
Cytoplasmic-mediated microRNA (miRNA) biogenesis. (a) Schematic of miR-124 insertion [depicted as noncoding RNA (ncRNA)] into the vesicular stomatitis virus (VSV) genome (N, nucleocapsid, P, phosphoprotein, M, matrix, G, glycoprotein, and L, large polymerase). ...

Next, we compared the production of miR-124 by VSV124 to that of SV124 and IAV124. Regardless of polarity, or site of virus replication, infection with recombinant RNA viruses resulted in robust miR-124 synthesis (Figure 2a). To determine whether processing of virus-derived miRNAs was dependent on the canonical cytoplasmic machinery, fibroblasts deficient in Dicer were infected with the miR-124 expressing RNA viruses. Consistent with our previous work,10 VSV-, SV-, and IAV-derived miR-124 production as well as endogenous synthesis of miR-93, were all completely abolished in the absence of Dicer (Figure 2a). Importantly, the abrogation of miR-124 was not due to a loss of virus transcription, as virus mRNA levels were similar between Dicer-deficient and wild-type fibroblasts (Figure 2b). Together these data demonstrate that an RNA virus of negative polarity can be engineered to produce a mature, dicer-dependent miRNA.

Figure 2
MicroRNA (miRNA) production by vesicular stomatitis virus (VSV) is Dicer-dependent. (a) Northern blot of RNA from wild-type (WT) or Dicer-deficient fibroblasts infected with control or miR-124 expressing viruses. RNA was probed for miR-124 (top) and miR-93 ...

Engineered cytoplasmic viruses deliver a high payload of miRNAs

In an effort to define the overall magnitude of cytoplasmic-derived miR-124 expression, we infected murine fibroblasts with each of the three virus vectors, and analyzed the small RNA fraction by deep sequencing (Figure 3a). Aligning captured small RNAs to the 583 nucleotide, virus-derived, pri-miR-124 yielded ~80,000–400,000 specific reads mapping to this transcript. Not surprisingly one of the most abundant species captured, mapped to miR-124 in response to each engineered RNA virus infection. Vector-derived miR-124 accumulated to levels as high as 2% of the total miRNAs profiled, reaching estimated concentrations ranging from 25,000 to 35,000 copies/cell (Figure 3a, Supplementary Table S1 and see Materials and Methods). As miR-124 expression is limited to the brain,15 levels from mock-infected cells represented <0.001% of the total miRNAs profiled (<15 copies/cell). This deep sequencing analysis suggests that delivery of miRNAs by cytoplasmic RNA viruses yields a high copy number and frequency of miRNAs on a per cell basis, which is critical for efficient PTS.17 In addition to the overall abundance of virus-derived miR-124, another striking feature of the small RNA profiling was the accumulation of star-strand RNA. Restricted to the cytoplasmic viruses, the amount of star strand captured by deep sequencing represented as much as 40% of the total RNA mapping to pri-miR-124 (Figure 3a). This unique activity elicited by cytoplasmic viruses was specific for miR-124, as star-strand accumulation for endogenous miRNAs, such as miR-93, and miR20a, or alterations in 5p-3p species, was not detected (Figure 3b and Supplementary Figure S1a,b).

Figure 3
Deep sequencing analysis of virus-derived miR-124. Deep sequencing analysis for miR-124 and miR-124 star (*) strand in mock, SV124, VSV124, and IAV124 infected murine embryonic fibroblasts. (a) Percent of miR-124 and miR-124* transcripts ...

We next corroborated our deep sequencing results by small RNA northern blot. As expected, cytoplasmic miR-124-producing viruses, demonstrated miR-124 star-strand production, albeit at a lower abundance than the mature strand (Figure 3c,d). To determine whether miR-124 star accumulation was a consequence of miRNA overexpression or an effect of cytoplasmic processing, we compared virus- and plasmid-derived miR-124 and miR-124 star synthesis. Utilizing a cell line conducive for high-transfection efficiency and transcript synthesis, we demonstrated that plasmid-derived miR-124 (p124) also showed evidence of miR-124 star accumulation (Figure 3c,d). However, even under these conditions, the levels of plasmid-derived miR-124 star were significantly lower than that observed from SV124 and VSV124, suggesting that accumulation, while partially due to an over abundance of pre-miR-124, may also be a defining characteristic of cytoplasmic processing of miRNAs.

Cytoplasmic-derived miR-124 associates with Ago2 to mediate PTS

While the high frequency and copy number of cytoplasmic-derived miRNAs suggests the ability to mediate PTS, the increased presence of miRNA star strand could be indicative of a lack of RISC loading and subsequent duplex separation. Therefore, we next determined whether virus-derived cytoplasmic miRNAs associated with Ago2, the most characterized member of the mammalian Argonaute family and only member that has retained slicing activity.1 Cells expressing epitope tagged -Ago2 or green fluorescent protein (-GFP), were mock-treated or infected with miR-124-expressing viruses, to ascertain whether Ago2 associated with miR-124 (Figure 4a). While virus-produced miR-124 was not detected by immunoprecipitation of infected, GFP-expressing cells, Ago2 associated with miR-124, regardless of intracellular origin (Figure 4a and Supplementary Figure S2). Furthermore, the levels of Ago2-associated endogenous miR-93 were not diminished as a result of robust synthesis of miR-124 (Figure 4a). However, given the potential saturation of Ago2, we sought to confirm that engineered RNA virus expression of miR-124 did not inhibit the function of other miRNAs. To this end, cells were cotransfected with miR-142 and a GFP reporter containing four miR-142 response elements in the 3′UTR. Consistent with previous studies18 over expression of miR-124 by all three virus vectors did not inhibit miR-142 repression of the GFP reporter. Together these data suggest that virtrons, and miR-124 expressed from IAV, do not inhibit the function of miRNAs and therefore are unlikely to cause additional toxicity.

Figure 4
Engineered viral microRNA (miRNA) is loaded into RNA-induced silencing complex (RISC) and mediates post-transcriptional silencing (PTS). (a) Immunoprecipitation (IP) of extract from 293T cells transfected with Flag-tagged Ago2 or green fluorescent protein ...

An additional concern with regards to off-target effects elicited by virtrons derives from the accumulation of miRNA star strand. The increased miRNA star-strand expression exhibited by both SV124 and VSV124 suggested the possibility that both the guide and passenger strand mediate PTS. To address this issue, cells expressing luciferase containing either the miR-124 or miR-124 star target sites in the 3′UTR were infected with miR-124-expressing viruses to assess PTS activity. As expected, all three engineered RNA viruses were capable of mediating significant repression of the miR-124 targeted luciferase reporter, demonstrating ~60–90% repression of the luciferase construct (Figure 4c). Furthermore, virtrons were also capable of significantly inhibiting luciferase containing the 3′UTR of Scp1, encoding endogenous miR-124 response elements15 (Supplementary Figure S3). However, we also observed a significant repression of the miR-124 star target, albeit at significantly reduced levels compared to miR-124 activity (Figure 4c). Given that miR-124 star-mediated PTS correlated to the relative levels between mature and star-strand accumulation (Figure 3c,d), it is likely that any off targeting, as a result of improper strand loading, could be further minimized by adjusting the thermodynamic properties of the miRNA duplex.19

In vivo production of cytoplasmic-derived miRNAs

Given the evidence for in vitro pri-miRNA cytoplasmic processing from diverse RNA sources, we next sought to determine whether this activity could be recapitulated in vivo. In an effort to compare virus-derived miRNA synthesis directly, independent of the innate antiviral response, we used mice deficient in type I interferon signaling to ensure equal levels between viral cohorts.20 To this end, interferon-α receptor I (Ifnar1) knockout mice were infected with miR-124-producing viruses to monitor miRNA synthesis following infection. Strikingly, recombinant viruses yielded high levels of miR-124 within lungs of infected animals in as early as 1 day postinfection (dpi) (Figure 5a).

Figure 5
RNA virus-derived production of microRNA (miRNA) in vivo. (a) Northern blot of RNA derived from Ifnar1−/− mice infected intranasally with Sindbis virus (SV), vesicular stomatitis virus (VSV), or influenza A virus (IAV)-expressing miR-124. ...

To further characterize the therapeutic potential of cytoplasmic RNA virus-derived miRNA synthesis, we focused on VSV-mediated delivery because of its broad tissue tropism and safety record in preclinical trials.21,22 To determine the tissues capable of supporting VSV-mediated synthesis of miRNAs, we again used Ifnar1 knockout mice as a tool to allow for systemic spread in the absence of cellular antiviral signaling. Following intravenous infection, we were able to visualize miR-124 expression by small RNA northern blot, in as early as 48 hours postinfection (hpi), in lung, spleen, liver, kidney, and heart which correlated with VSV transcript levels (Figure 5b and Supplementary Figure S4). Given that tropism can also be controlled using cell-specific miRNA targeting technology,17,23–26 in combination with VSV's broad tissue distribution, this vector could be further modified to generate tissue-specific therapeutic tools.

Functional delivery of virtrons in vivo

Given the abundance of vector-derived miR-124 in Ifnar1 knockout mice, we next sought to determine whether these levels could be recapitulated in an immune-competent animal. As such, wild-type mice were infected i.n. with VSVscbl or VSV124 and RNA from whole lung was probed for VSV leader RNA (LRNA), a small noncoding RNA naturally produced by the virus during replication. In both infections, VSV LRNA was detected within 1 dpi. Furthermore, VSV124 LRNA levels were sustained until 4 dpi at which time virus levels markedly decreased (Figure 6a). To determine whether VSV-derived miR-124 levels persisted following virus clearance, or demonstrated a precipitous drop correlating with virus replication, we probed total lung for miR-124 and compared this to LRNA expression. Despite the loss of LRNA at 5 dpi, VSV-derived miR-124 was maintained at high relative levels, suggesting that virtrons persist even during vector clearance (Figure 6a).

Figure 6
Functional delivery of virtrons in vivo. (a) Northern blot of RNA from wild-type (WT) mice intranasally infected with either VSVscbl or VSV124. RNA was extracted at 0–5 dpi and probed for miR-124 (top), vesicular stomatitis virus (VSV) leader ...

As VSV124 was capable of delivering high levels of miR-124 in wild-type mice, we next sought to determine whether the miRNA delivered was capable of mediating PTS and modulating the cellular transcriptome. To analyze in vivo functionality of virtrons, we performed quantitative reverse transcriptase-PCR on total lung from mice infected with VSVscbl or VSV124 at 24 and 72 hpi (Figure 6b). To this end, we analyzed expression levels of polypyrimidine tract-binding protein 1 (Ptbp1), a miR-124 target, validated by three independent groups, including in vivo endogenous data,15 overexpression studies27 and by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS CLIP).28 As a control, we compared Ptbp1 levels to Tnfa, a transcript known to be induced during VSV infection but not implicated in miR-124 PTS.27,29 As expected, VSV infections increased Tnfa levels over naive mice, demonstrating no significant difference between virus cohorts at 24 and 72 hpi (Figure 6b), time points where VSV-derived 124 expression is maintained at robust levels (Figure 6a). In contrast, while VSVscbl infection also resulted in a 30-fold induction of Ptbp1, infection with VSV124, resulted in less than a fivefold increase (Figure 6b). Given that this analysis includes uninfected pulmonary cells, in addition to the fact that miRNAs result in only modest transcriptional silencing,30,31 the measurable decrease in Ptbp1 transcripts strongly suggests miR-124 to be functional in vivo. Taken together, these data suggest that miRNA delivery by a cytoplasmic RNA virus is capable of mediating PTS and further validates this as a novel vector to deliver therapeutic miRNAs.

Discussion

The mechanism underlying production of miRNA biogenesis from a cytoplasmic virus remains unclear, however, the data presented herein validates this activity as a bona fide molecular process. The ability of cytoplasmic RNA viruses to generate functional miRNAs in vivo suggests that this process is not an artifact of rapidly dividing transformed cells as has recently been suggested.13 As VSV-mediated synthesis of pri-miR-124 would mimic an endogenous mRNA, containing a 5′cap and a polyadenylated tail, its transport back into the nucleus of primary cells would be highly improbable. It then follows that the cell must contain cytoplasmic small RNA biogenesis components responsible for this activity or, alternatively, that formation of the hairpin recruits components of the canonical machinery into the cytoplasm. Consequently, these observations imply the cell may produce endogenous small RNAs that are processed independently of nuclear activities.

Deep sequencing analysis of these vectors defined intriguing characteristics of cytoplasmic-derived mature miRNAs. This analysis revealed that virtron levels could range from an estimated 28,000–35,000 copies/cell for SV124 and VSV124, respectively. A distinct feature of these virtrons is an increase in the amount of miRNA star strand. This phenotype, along with virtron DGCR8-independency, is another characteristic shared with mirtrons, noncanonical miRNAs derived from debranched lariats and originally described in Drosophila.10,32,33 These data suggest that extensive star-strand accumulation may be a consequence of noncanonical biogenesis. Alternatively, overexpression of miRNAs could also lead to RISC saturation and increased duplex separation, thereby increasing the levels of detectable miRNA star. However, overexpression of miR-124 by plasmid resulted in detectable, but reduced levels of star strand, suggesting that over expression of miRNA is not solely responsible for the observed accumulation and that this may represent a defining characteristic of both mirtron and virtron processing. Furthermore, the increased miRNA star strand was capable of repressing a corresponding target reporter, illustrating potential off-target adverse effects. While potentially problematic, this activity could be mitigated by diminishing star stand accumulation through the alteration of 5′ thermostability, or changing the star-strand sequence to prevent unwanted targeting.

The ability to deliver miRNAs or small interfering RNAs (siRNAs) in vivo has been a major hurdle in harnessing the power of RNA interference. Here, we describe the utilization of cytoplasmic vectors to deliver functional miRNAs to a wide array of tissues. While the mechanism underlying cytoplasmic processing remains unclear, the in vivo functionality of these vector-derived small RNA payloads provides a novel strategy for future therapeutics. Any of the viruses described in this study could be utilized for small RNA delivery in vivo, however, VSV may represent the most malleable vector. VSV has already proven to be safe in preclinical toxicology studies,21 and has been associated with only minimal disease in a small fraction of the veterinarian or agricultural workers that have been infected.34 Furthermore, the reverse genetics system for VSV permits for additional modifications to the virus, allowing one to control levels of virus replication and/or tropism.16,25,26,35–37 Similarly, RNA virus-like particles could be developed into a novel therapeutic platform to deliver miRNAs in vivo. This platform would not only apply to the delivery of endogenous miRNAs, but could also be extended to artificial miRNAs, in which the stem can be redesigned to generate siRNAs against a transcript of interest.38 Currently, one of the major hurdles to therapeutic gene silencing by small duplex RNA (short hairpin RNA , siRNA, miRNA, or artificial miRNA) has been delivery. Toxicity issues stemming from saturation of the nuclear miRNA biogenesis and export machinery have plagued delivery by DNA viruses.39,40 Additionally, extended high expression of exogenous short hairpin RNAs by these same viruses result in Ago2 saturation and increased liver toxicity.41 Fortunately, cytoplasmic virus-based vectors bypass these constraints and provide transient expression of a given small RNA. As virus-derived small RNAs persist beyond infection, an attribute of their long half-life,42 these vectors would be most suited to treat diseases which, although possibly chronic, manifest acute phenotypes that do not generally exceed 7 days. Therapeutic applications for these vectors would likely exploit artificial miRNAs that produce perfectly complementary siRNAs through the miRNA pathway, thereby reducing bottlenecking at the level of nuclear export and/or RISC saturation. In this way, these vectors may be useful in the treatment of virus infection, or in the enhancement of virus oncolytics.22,43 Another intriguing aspect of these findings is the development of a new tool to differentiate stem cells ex vivo through the delivery of miRNAs44 or as a tool to discover novel virus–host interactions. In the latter example, viruses could be engineered to express a library of artificial miRNAs that would only be delivered to infected cells, permitting evolutionary-selection to parse out essential host restriction factors as an alternative approach to classic siRNA screens which have elucidated numerous antiviral factors.45 In all, these data suggest that generation of miRNAs within the cytoplasm is a bona fide molecular process and that this activity can be exploited for the development of future small RNA-based therapeutics.

Materials and Methods

Small RNA northern blot analyses and deep sequencing. Small RNA northern blots and probe labeling were performed as previously described.12,46 Probes used include: anti-miR-124: 5′-TGGCATTCACCGCGTGCCTT AA-3′, anti-miR-93: 5′-CTACCTGCACGAACAGCACTT TG-3′, anti- U6: 5′-GCCATGCTAATCTTCTCTGTATC-3′, anti VSV LRNA 5′-GTTTCTCCTGAGCCTTTTAATGATAATAATGGTTT GTTTGTCTTCGT -3′. For deep sequencing analysis, miR-124-specific small RNA libraries were generated as previously described.47 Briefly, total RNA from SV, VSV, and IAV expressing miR-124 infected murine embryonic fibroblast samples was extracted 16 hpi using Trizol as per the manufactures instructions. Twenty micrograms of total RNA were separated on a 12% denaturing Tris–urea gel alongside a labeled Decade marker (Ambion, Austin, TX). Small RNA species between 15 and 30 nucleotides were then isolated, purified, and amplified as previously described.10 The resulting small RNA was ligated to a 5′-adenylated 3′ adapter oligonucleotide (5′AppCTGTAGGCACCATCAATdideoxyC-3′; Integrated DNA Technologies, Coralville, IA) using the Rnl2 AirTM Ligase (BIOO Scientific, Austin, TX) in the absence of ATP. The ligation product was separated from the unligated adapter by gel purification, and ligated to the 5′ adapter RNA oligonucleotide using T4 RNA ligase (NEB). Following gel isolation, the ligation product was reverse transcribed, PCR amplified (21 cycles) and purified by agarose-based gel electrophoresis. The small RNA library quality was assessed on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). Each small RNA library was sequenced on an Illumina Genome Analyzer II Platform. The sequences were mapped as contigs to the pre-miRNA of miR-93 and miR-124 as listed on miRBase (gro.esabrim.www). Copy numbers were estimated on miR-93 and Mcm7 expression which is expressed at 1.00 × 104–1.00 × 105 molecules/cell in murine fibroblasts.48

Vector design for cytoplasmic miR-124 synthesis. Generation of Sindbis and IAVs-expressing miR-124 have been described elsewhere.10,14 VSV expressing the pri-miR-124 genomic segment (chr3:17,695,454-17,696,037) was generated and rescued as previously described.49 GFPmiR-124 was generated from pEGFP-C1 (Clontech, Mountain View, CA).

Cell culture and PTS. Dicer1 knockout cells used in this study are described elsewhere.23,50 Baby hamster kidney cells were transfected with miR-142t_GFP alone or in conjunction with miR-142 and 2 hpt cells were infected with control or miR-124 expressing VSV, SV, and IAV at multiplicity of infection of 1, 1, and 3, respectively. Sixteen hpi cells were run on a FACS calibur (BD, Franklin Lakes, NJ) and live cells analyzed for GFP expression by Flowjo (Treestar, Ashland, OR). Baby hamster kidney cells were transfected with either Gaussian luciferase containing 4 tandem repeats of the miR-124 or miR-124* target sites. Cells were cotransfected with untargeted firefly luciferase and levels of targeted Gaussian luciferase were normalized to the untargeted control. Cells were infected with control or miR-124 expressing VSV, SV, and IAV at multiplicity of infection of 1, 3, and 5, respectively. Luciferase expression from 124 expressing virus was compared to control virus infection. As a positive control cells were also transfected with p124 and knockdown calculated compared to empty vector control. Percent target repression was normalized to control transfection or virus infection at 24, 18, 24, and 24 hpi for p124, VSV, SV, and IAV, respectively (see Supplementary Materials and Methods). P values for Luc_124t knockdown are as follows: p124 P = 0.0004, SV124 P = 0.001, VSV124 P = 0.0001, and IAV P = 0.0022 and for Luc_124t* are: p124 P = 0.735, SV124 P = 0.04, VSV124 P = 0.0009, and IAV P = 0.12.

In vivo infections. BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). Ifnar1−/− mice were a kind gift from Thomas Moran, Mount Sinai School of Medicine. Ifnar1−/− mice were anesthetized with isofluorane and infected intravenous with 2 × 105 plaque-forming unit (pfu) SV124 or i.n. with 2 × 107 pfu of VSV124 or 1 × 107 pfu of IAV124. Lungs were removed 1 dpi for VSV124 and 2 dpi for SV124 and IAV124. VSV124 was also injected intravenous at 1 × 104 pfu. BALB/c mice were infected i.n with 4 × 107 pfu of VSVscbl or VSV124 and lungs were removed on 1–5 dpi. All experiments involving animals were done in accordance with Mount Sinai School of Medicine Institutional Animal Care and Use Committee.

Immunoprecipitations and quantitative reverse transcriptase-PCR. Immunoprecipations were performed in 293 cells. Cells were transfected with 12 µg of either flag-tagged Ago2 or flag-tagged GFP (addgene Cat #'s 19888 and 22612, respectively) and subsequently infected with either wild-type or miR-124 expressing SV, VSV, or IAV. Protein extracts were harvested 12 hpi and were immunoprecipitated with Protein-G-PLUS agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and 10 µg of anti-Flag (Sigma, St Louis, MO) for 12 hours at 4 °C. Beads were washed and RNA extracted with TRIzol (Invitrogen, Carlsbad, CA). Quantitative reverse transcriptase-PCR of complementary DNA samples was performed using KAPA SYBR FAST qPRC Master Mix (KAPA Biosystems, Boston, MA). PCR were performed on a Mastercycler ep realplex (Eppendorf). Actin or murine tubulin were used as the endogenous housekeeping genes and Delta delta cycle threshold (ΔΔCT) values were calculated with replicates over actin or tubulin. Values represent the fold change over mock-infected samples.

Statistical analysis. Statistical analysis was performed on indicated samples using a two-tailed, unpaired Students-t test. Data are considered significant if P value is <0.005.

SUPPLEMENTARY MATERIAL
Figure S1. Endogenous miRNA:miRNA* levels during RNA virus-derived miRNA synthesis.
Figure S2. Protein levels from Ago2 association studies.
Figure S3. RNA virus-derived miR-124 repression of an endogenous 3'UTR reporter.
Figure S4. In vivo virus transcript levels correlate with mature miR-124 levels.
Table S1. Deep sequencing synopsis of miR-124-producing viruses.
Materials and Methods.

Acknowledgments

This material is based upon work supported in part by the US Army Research Laboratory and the US Army Research Office under grant number W911NF-07-R-0003-4. R.A.L. and A.M.P. are supported by the NYU-MSSM Mechanisms of Virus–Host Interactions National Institutes of Health T32 training grant (no. AI007647-09). B.R.tO is supported in part by the Pew Charitable Trust and the Burroughs Wellcome Fund. We would additionally like to thank Alexander Tarakhovsky (Rockefeller University) and Donal O'Carroll (EMBL, Monterotondo, Italy) for Dicer1−/− fibroblasts, and the members of the MSSM Microbiology department for helpful discussions.

Supplementary Material

Figure S1.

Endogenous miRNA:miRNA* levels during RNA virus-derived miRNA synthesis.

Figure S2.

Protein levels from Ago2 association studies.

Figure S3.

RNA virus-derived miR-124 repression of an endogenous 3'UTR reporter.

Figure S4.

In vivo virus transcript levels correlate with mature miR-124 levels.

Table S1.

Deep sequencing synopsis of miR-124-producing viruses.

Materials and Methods.

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