Small non-coding RNAs have emerged as key modulators of viral infection. However, with the exception of hepatitis C virus, which requires the liver-specific microRNA (miRNA)-122, the interactions of RNA viruses with host miRNAs remain poorly characterized. Here, we used crosslinking immunoprecipitation (CLIP) of the Argonaute (AGO) proteins to characterize strengths and specificities of miRNA interactions in the context of 15 different RNA virus infections, including several clinically relevant pathogens. Notably, replication of pestiviruses, a major threat to milk and meat industries, critically depended on the interaction of cellular miR-17 and let-7 with the viral 3′ UTR. Unlike canonical miRNA interactions, miR-17 and let-7 binding enhanced pestivirus translation and RNA stability. miR-17 sequestration by pestiviruses conferred reduced AGO binding and functional de-repression of cellular miR-17 targets, thereby altering the host transcriptome. These findings generalize the concept of RNA virus dependence on cellular miRNAs and connect virus-induced miRNA sequestration to host transcriptome regulation.

In the current study, we used AGO-CLIP and analysis of miRNA-target chimeras to examine a broad panel of 15 different RNA viruses for miRNA interactions in mammalian cells. Of particular interest were uncovered interactions between miR-17 and let-7 and the 3′ UTR of bovine viral diarrhea virus (BVDV), which were critical for viral replication. BVDV and classical swine fever virus (CSFV) are members of the Pestivirus genus, an important group of animal pathogens distantly related to HCV within the Flaviviridae family. By examining host transcriptomes in vitro and ex vivo, we observed reduced AGO binding and functional mRNA de-repression of cellular miR-17 targets but not let-7 targets during pestivirus infection. This virus-host interaction paradigm provides a mechanism by which viruses can selectively regulate hundreds of cellular genes with minimal investment of genomic information.

Studies of miRNA action were enhanced by AGO-CLIP methods (), which were used to characterize miRNA expression and regulation for several DNA viruses (). More recently, we generated small RNA binding landscapes for HCV RNA, confirming the 5′ UTR miR-122 interaction and unexpectedly demonstrating that sequestration of miR-122 by HCV RNA leads to transcriptome-wide de-repression of cellular miR-122 targets (). A major drawback of standard AGO-CLIP methods is the inability to link miRNAs directly to their targets. miRNA-target chimeras produced through direct ligation allow unambiguous identification of such interactions (). Through covalent ligation of endogenous argonaute-bound RNAs (CLEAR)-CLIP on endogenous AGO complexes, we recently improved target identification efficiency ().

Direct and indirect interactions with host miRNAs play important roles for DNA viruses, many of which encode their own miRNAs (). For RNA viruses, interactions with host miRNAs remain poorly characterized with the exception of HCV, which requires the liver-specific miR-122 (). The discovery of interactions between miR-122 and the HCV 5′ UTR () at two binding sites stimulating viral replication was unexpected, as miRNAs typically interact with the 3′ UTRs of mRNAs to destabilize transcripts or repress translation (). The HCV/miR-122 interaction has proven promising as therapeutic target (). In contrast, Eastern equine encephalitis virus (EEEV) is an example of canonical miRNA action, in which conserved miR-142-3p sites limit viral replication and thereby activation of innate immunity specifically in hematopoietic cells, a mechanism that enhances neuropathogenesis ().

Hepatitis C virus and human miR-122: insights from the bench to the clinic.

Taken together, these results demonstrated that pestiviruses in vitro and ex vivo induce a miR-17-specific de-repression, thereby regulating hundreds of cellular targets.

Lastly, to evaluate how the aggregate gene regulation by sponging may influence pestiviral replication, we decoupled the direct miR-17 requirement from sponging by infecting miR-17p3,4 trans-complemented MDBK cells with BVDV-m17p3,4 and mimicking sponging using tinyLNA-17 ( Figure 7 I). Observing no change in virus production, we concluded that pestiviral miR-17 sponging does not appear to directly influence infection kinetics in this context.

We investigated the miRNA sequestration for another pestivirus by performing mRNA-seq 8 and 20 hr after CSFV infection of porcine monocyte-derived macrophages ex vivo. In both unpolarized and interferon-γ-polarized macrophages, we observed specific, time-dependent de-repression of mRNAs harboring miR-17 sites in the 3′ UTR ( Figures 7 F, 7G, S7 K, and S7L). Among genes expressed in unpolarized macrophages with a miR-17 8-mer in the 3′ UTR, 32 were significantly upregulated upon CSFV infection ( Table S5 ). The de-repression of miR-17 targets induced by BVDV and CSFV (both high and low virulence strains) was confirmed at the protein level using psiCHECK-2 reporters harboring miR-17 seed sites in the 3′ UTR ( Figure 7 H).

Along these lines, we reasoned that the BVDV miR-17 sponge effect should result in specific de-repression of cellular miR-17 targets, measurable by mRNA-seq. Indeed, mRNAs harboring a CLIP-guided miR-17 site in their 3′ UTR were significantly de-repressed compared to those having a site for let-7, miR-15, or the top 10 miRNAs in MDBK cells ( Figure 7 E and S7 H and Table S4 ). Among mRNAs with a CLIP-supported miR-17 8-mer in their 3′ UTR, 19 were significantly upregulated during BVDV infection ( Table S5 ). Using alternative parameters to identify miR-17 sponge-regulated genes, 47 mRNAs had reduced AGO binding at 3′ UTR miR-17 6-8-mer sites and were significantly upregulated during BVDV infection ( Table S5 ). Excess amounts of miR-17p3,4 dramatically repressed an arbitrary set of mRNAs carrying the corresponding seed site in their 3′ UTRs ( Figure S7 I), and BVDV-m17p3,4 mutants were not able to induce a sponge effect under these conditions ( Figure S7 J). Thus, the BVDV miR-17 sponge effect is functional but only within a certain range of miRNA abundance.

Given specific interactions between BVDV and miR-17 and let-7, we analyzed AGO binding on the host transcriptome during infection ( Figure S7 B). We observed significantly reduced global AGO binding on 3′ UTR and coding sequence (CDS) targets of miR-17 but not let-7 when compared to targets of the miR-15 family, the top 10 miRNAs, or all targets ( Figures 7 B and S7 C–S7F and Table S4 ). A similar but stronger effect was observed when comparing tinyLNA-17 to mock-treated cells ( Figures 7 C and S7 G and Table S4 ). Furthermore, we observed that BVDV RNA genomes can approach miR-17 copy numbers ( Figure 4 F) and that the abundance of miR-17 family members available for regulation is decreased upon infection ( Figure 7 D). Taken together, these data suggest that miR-17 but not let-7 can be functionally inhibited by BVDV RNA.

We reasoned that the number of miRNA chimeric reads mapping to the virus versus the host would be a direct way to evaluate specific miRNA sequestration during infection. For HCV, around 50% of miR-122 interactions mapped to the virus ( Figure 7 A), consistent with our modeling estimates of the available miR-122 pool during infection (). For BVDV, around 40% of miR-17 interactions mapped to the virus, suggesting an analogous sponge effect. For let-7, this number was <10% due to the high abundance of this miRNA family ( Figure S7 A and Table S3 ). The sponge effects of HCV and BVDV were specific to miR-122 and miR-17, respectively; in contrast, SINV and CHIKV sequestered a broad range of miRNAs consistent with their general AGO sequestration ( Figures 4 B and 7 A).

(I) Virus production after infection of miR-17p3,4 trans-complemented (10 nM) MDBK cells with BVDVcp-m17p3,4 ± tinyLNA-17. Mean ± SD is shown (H and I). See also Figure S7 and Tables S3 S4 , and S5

(H) Reporter validation of target de-repression by BVDV in MDBK and CSFV in SK-6 cells. Schematics of reporters are as in Figure 6

(F and G) CDF plots as in (E) but for CSFV vEy-37-infected unpolarized porcine monocyte-derived macrophages 8 hr (F) and 20 hr (G) post infection, from triplicate experiments.

(E) CDF plot for BVDV infection of MDBK cells as in (B) but measuring target expression via mRNA-seq, from duplicate experiments.

(D) Volcano plot for significant changes to AGO/miRNA profiles after infection with BVDV. miRNAs with a significant change in abundance are shown in blue and those of the miR-17 family or the miR-17-92 polycistron in boldface.

(B) Cumulative density function (CDF) of the log 2 -fold change in CLIP binding between BVDV infected and uninfected cells for all 3′ UTR clusters containing indicated 8-mer seeds, from quadruplicate experiments. “Top 10” refers to the ten most abundant CLIP-derived AGO-bound miRNAs. Two-sided K-S test p values of difference to “All” are shown.

(A) Percentage of virus-derived versus total chimeras shown for selected miRNAs as a direct quantification of miRNA sponge effects. A schematic of specific versus general miRNA/AGO sequestration is depicted to the left.

To assess the effect of miR-17 on intracellular BVDV RNA stability, we halted ongoing BVDV replication in MDBK cells in the presence or absence of tinyLNA-17 or excess miR-17. Measuring the relative level of BVDV RNA over time, we observed a small, non-significant stabilization by miR-17 and de-stabilization by tinyLNA-17 ( Figure 6 E). When BVDV-pol(-) RNA was transfected, excess miR-17 provided a small, non-significant protection from degradation ( Figure 6 F). A larger and significant effect was seen when BVDV-pol(-)m17p3,4 was stabilized by miR-17p3,4 ( Figure 6 G). A similar effect was not observed for BVDV-pol(-)let-7p7,8 ( Figure 6 H). These results indicate that the miR-17 but not let-7 interaction plays a small protective role in viral RNA degradation.

In a further attempt to analyze non-canonical binding of let-7, we measured translation from bicistronic BVDV-m17p7,8 reporters. Trans-complementation with miR-17p7,8, but not let-7p17,18, rescued translation ( Figure S6 G). Further, rescue of translation depended on an intact terminal stem loop and was not affected by swapping the stem base pairs ( Figure S6 G). Thus, putative non-canonical binding does not appear important for translation.

Because these results differ from canonical miRNA repression of cellular mRNAs, we confirmed the positive role for miR-17 on translation in context of monocistronic reporters containing the exact BVDV ends ( Figure 6 C). In contrast, in a capped, poly(A)-tailed mRNA-like context, seed site mutations led to increased translation. A combined let-7/miR-17 BVDV 3′ UTR mutant relieved translational repression to the greatest extent ( Figure 6 D). Thus, it appears that the positive role of let-7 and miR-17 on translation occurs specifically in context of a non-capped, non-poly(A)-tailed, IRES-containing RNA, like those of pestiviruses.

For HCV, miR-122 modestly stimulates viral translation () and protects the uncapped RNA genome from degradation (). Using bicistronic BVDV reporters, translation was non-significantly decreased after mutating the let-7 site and decreased to ∼70% and ∼50% after mutation of the miR-17 alone or in combination with let-7 ( Figure 6 A). Trans-complementation with high levels of the corresponding miRNAs rescued translation to ≥100%. Inhibition of miR-17 by tinyLNA-17 reduced translation to ∼80% ( Figure 6 B). Translation was strongly inhibited by deleting the 3′ UTR terminal stem loop. Retaining the terminal 19 nt of the 3′ UTR partially reversed this phenotype ( Figure 6 B). This suggested that both the terminal 3′ UTR sequence and the miRNA interactions positively regulate BVDV translation, with miR-17 playing a larger role than let-7.

(G and H) RNA decay assays as in (F) but for seed site mutants ± corresponding miRNA complementation. Mean ± SEM is shown. See also Figure S6

(E and F) Effect of miRNAs on BVDV RNA stability. Decay of BVDV RNA was measured ± tinyLNA-17 (0.3 μM) or miR-17 (50 nM) after blocking active BVDV replication using 2′CMA (E) or after transfection of replication-deficient pol(-)BVDV RNA (F).

(D) Translation measured from RNAs harboring the BVDV 3′ UTR and/or miRNA seed sites in the capped, poly(A)-tailed context of the psiCHECK-2 system. In the schematics, caps are black circles, miRNA sites are squares, promoters are angled arrows, and poly(A)-tails are AAAs.

(C) Translation as in (B) but measured from monocistronic reporters with the exact BVDV termini.

(B) Translation as in (A) measured after treatment with tinyLNA-17 (0.3 μM) or miR-17 (50 nM) or after modifications to the 3′ UTR as indicated in the schematic below.

(A) BVDV-driven translation was measured from bicistronic reporters after introduction of let-7 or miR-17 seed site mutations and after trans-complementation with miRNA mimics (50 nM). Differences from the wt BVDV 3' UTR was evaluated by ANOVA. ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001.

The let-7 and miR-17 seed sites are highly conserved among pestiviruses ( Figure 5 H), suggesting that pestiviruses generally bind and depend on these host miRNAs. To test this hypothesis, we infected SK-6 cells with CSFV and observed decreased virus production ( Figure 5 I) after functional inhibition of miR-17 by tinyLNA-17 ( Figure S6 A). A CSFV-m17p3,4 mutant had reduced focus size and virus production after electroporation. This phenotype could be rescued by trans-complementation using miR-17p3,4 ( Figures 5 J, S6 B, and S6C). We further established SK-6 cells stably expressing miR-17p3,4 by lentiviral transduction and fluorescent sorting ( Figures S6 D and S6E), which, in contrast to parental cells, efficiently propagated WT as well as m17p3,4 mutant CSFV ( Figure 5 K). Thus, dependence on host miRNAs appears to be a general phenomenon in pestivirus replication. We note that the near-universal tissue tropism of pestiviruses () is congruent with the ubiquitous expression of miR-17 and let-7 across a range of tissue types () and cell lines studied here ( Figure S6 F).

To evaluate the importance of weaker BVDV-miRNA interactions, we analyzed miR-92a-3p as an example, for which predictions allowed both canonical and auxiliary binding ( Figure S2 A). We did not observe any effect on BVDV replication or virus production from inhibition of this miRNA ( Figures S5 G and S5H), arguing against importance of minor miRNA interactions.

If the putative non-canonical binding of let-7 is important for replication, predictions suggest that a BVDV-m17p7,8 mutant would require miR-17p7,8 as well as trans-complementation with let-7-p17,18. However, BVDV-m17p7,8 virus production was unaffected by the presence of let-7i-p17,18 ( Figure S5 E). Further, a BVDV-m17p7,8 mutant with a swap of two base pairs at the base of the terminal stem loop was viable ( Figure S5 E), arguing against binding of let-7 to an open conformation of the stem loop ( Figure S4 C). These data argue against a critical role for non-canonical let-7 interactions.

The let-7 family is highly abundant in MDBK cells and could be inhibited only to ∼50% using tinyLNAs ( Figure S5 F). BVDV replication in cells partially devoid of let-7 activity was slightly decreased with significant difference only at 24 hr ( Figure 5 F). BVDV p3,4 mutants of the canonical let-7 site were highly attenuated in culture and could be rescued by let-7i-p3,4 ( Figure 5 G). This suggested that both miR-17 and let-7 were critical for BVDV replication.

Using mutated miRNA mimics, we tested whether the BVDV/miR-17 interaction required auxiliary base pairing, as was observed for HCV/miR-122 (). This was not the case, since miR-17p3,4,15,16 mimic rescued BVDV-m17p3,4 virus production to a similar extent as miR-17p3,4. Virus production could not be rescued by the miR-17p3,4,5,15,16 mimic with an added mismatch in the seed ( Figure 5 E).

Masking the 5′ terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex.

Following electroporation of MDBK cells with BVDV cp and ncp mutant RNAs that could no longer interact with miR-17 (m17p3,4 or p7,8 mutants), no virus production or cell death (cp variant) was observed ( Figures 5 D and S5 C–S5E). In one experiment, delayed virus production occurred; however, sequencing revealed reversion of the p3 site from day 5 post electroporation and complete reversion to wild-type (WT) 5 days after passage to naive cells. Trans-complementation with miR-17p3,4 or miR17p7,8 completely rescued virus production ( Figures 5 D and S5 C–S5E), and with increasing concentrations BVDV replication reached a plateau ( Figure S5 D).

To determine the functional importance of the miR-17 interaction during BVDV infection, we inhibited the miR-17 family in MDBK cells using tinyLNA-17 () ( Figure S5 A). For both BVDV biotypes, tinyLNA-17 led to significant attenuation of replication and virus production ( Figures 5 A and 5B ). This inhibition was reproducible in BT cells ( Figure S5 B) and occurred in a dose-dependent manner ( Figure 5 C), suggesting a therapeutic antiviral action similar to that of miR-122 inhibitors for HCV ().

(K) Virus production after CSFV WT or m17p3,4 RNA transfection of SK-6 cells transduced with lentiviruses expressing miR-17 or miR-17p3,4 or with the lentivirus backbone (puro, ctr). Mean ± SEM (A–C) or ± SD (D–K) is shown. See also Figures S5 and S6

(J) Dose response of CSFV WT or an m17p3,4 mutant 48 hr after transfection of SK-6 cells with miR-17p3,4.

(H) Conservation logo plot showing relative frequency of bases at every position in the 3′ end of BVDV (31 sequences), CSFV (68 sequences), and border disease virus (BDV, 7 sequences) downstream of the AT-rich region. Canonical and non-canonical miR-17 and let-7 sites are indicated. For BVDV, some strains (incl. NADL used in this study) carry a semi-conserved duplication (red line).

(E) Virus production as in (D) but with trans-complementing the m17p3,4 mutant with miR-17p3,4, miR-17p3,4,15,16, or miR-17p3,4,5,15,16.

(A and B) Replication and virus production after infection of MDBK cells with BVDVcp (A) or BVDVncp (B) (MOI = 0.1) ± tinyLNA-17 (0.3 μM)

Thus, AGO-CLIP and miRNA chimeras identified important miRNA interactions for BVDV, confirmed the previously characterized HBV/miR-15 interaction, and mapped miRNA binding landscapes for important picornaviruses and alphaviruses.

For the alphaviruses, we observed one particularly dominant interaction between the SINV E2 region and miR-92a-3p. Targeting this miRNA with a locked nucleic acid (LNA), however, had no effect on SINV replication or virus production ( Figures S4 D and S4E). These results are congruent with reporter gene assays showing that the predicted non-canonical binding of the miR-92a-3p 3′ region, as predicted for SINV ( Figure S2 D), did not functionally regulate reporter gene expression ( Figure S4 E).

To assess miRNA binding in different cellular contexts, we compared AGO binding and miRNA chimera profiles for CHIKV and CHIKV-GFP in Huh-7 hepatoma cells and STAT1fibroblasts and observed similar ASC and clustering values ( Figures 4 J and 4K). Overall binding landscapes were similar, including chimera-supported interactions with miR-21 and miR-181. As expected, interactions with miR-122 were observed only in Huh-7 cells and with let-7 only in fibroblasts, reflecting the miRNA composition of these cell types ( Figure S3 C).

miRNA chimera data were also interpretable for PV, CVB, SINV, CHIKV, and VEEV ( Figures 2 C, 2D, S2 C, and S2D). These viruses replicate to very high RNA copy numbers and had extensive AGO binding. To mitigate noise from non-specific binding, we analyzed AGO binding at early time points for CVB and SINV. Although the percentage of viral AGO binding increased over time, the ASC decreased due to the dramatic increase in viral RNA content ( Figure 4 H). AGO binding peaks generally remained at the same position and increased in read depth over time, although for SINV binding accumulated across the entire genome ( Figures 4 I, S3 A, and S3B). Taken together, these data suggested that the extent of AGO binding on CVB and SINV RNA scales with RNA abundance, as does accumulation of non-specific binding.

Several direct miRNA interactions were previously suggested for HBV (). The largest specific peak in our dataset overlapped with a conserved miR-15 site ( Figure 2 B). The HBV miR-15 interaction was confirmed using target-CLIP and chimera data; a non-conserved, non-canonical miR-320a interaction may also contribute to AGO binding at this site ( Figure S2 B). These data confirmed previous observations that HBV can bind miR-15/16 at this site, possibly to downregulate this miRNA family and indirectly prevent apoptosis (). A smaller peak overlapped a previously suggested miR-17 binding site (), although no chimeras were present to support this. In Huh-7.5 cells, we saw no evidence for binding of miR-122 or other miRNAs at previously suggested sites.

To assay BVDV-miRNA kinetics, we followed MDBK cells electroporated with BVDVcp or a replication deficient pol(-) mutant over time ( Figure 4 F). In contrast to observations for HCV and miR-122 (), AGO binding to the BVDV 3′ UTR was observed only at late time points and never on pol(-) mutant RNA ( Figure 4 G).

For BVDV, chimera analysis revealed that the strong AGO binding on the 3′ UTR was due to members of the let-7 and miR-17 families. The let-7 and miR-17 chimera peaks overlapped each other and canonical seed sites for both miRNAs (defined in), which were flanked by crosslink-induced mutation sites (CIMS;) providing nucleotide resolution of AGO binding ( Figures 2 A and 4 D). An additional let-7 peak suggested potential non-canonical let-7 binding overlapping the miR-17 site. Chimera-specific CIMS analysis supported the canonical miR-17 site and the non-canonical let-7 site ( Figure 4 D). Let-7 and miR-17 binding constituted >50% of the miRNA binding on BVDV and >80% of the binding on its 3′ UTR ( Figure 2 A). Relative to other miR-17 family members expressed in MDBK cells, miR-93 binding to the BVDV genome was enriched compared to binding to cellular RNA. Among expressed let-7 members, let-7i was enriched relative to 7a on the canonical BVDV site; on the putative non-canonical site, only 7b and 7i bound ( Figure 4 E). These findings corresponded to energetically favorable binding of miR-93 and let-7b and 7i ( Figure S4 C). The read depth of other peaks across the BVDV ORF constituted only 3%–4% of the 3′ UTR peak and included let-7, miR-125b, miR-193a, and miR-92b-3p ( Figures 2 A and S2 A).

To quantitatively describe the extent of AGO binding to viral versus host RNA, we calculated the viral CLIP read fraction normalized to total read depth. We further normalized by the fraction of viral RNA over the amount of total RNA determined by RT-qPCR ( Figure S4 A) and used this to define the AGO sequestration coefficient (ASC; Figure 4 A). Whereas most viruses bound less than a few percent of the AGO pool and did not specifically sequester AGO, two outgroups were identified; those that bound significant proportions of the AGO pool due to extensive viral RNA amounts (low ASC, primarily alphaviruses, Togaviridae), and those that bound only a few percent of the AGO pool but were highly enriched for AGO binding relative to their replication level (high ASC, HCV, and BVDV; Figure 4 B). For HCV and BVDV, AGO/miRNA binding further was highly clustered with up to 40% of reads constituting the single largest binding peak ( Figures 4 C and S4 B). Although these parameters alone do not suffice for identifying interactions of biological significance, strong, specific, and perhaps essential AGO interactions would be expected for viruses with high ASC and clustering, which was the case for HCV, for BVDV, and, to a lesser degree, for HBV.

(K) AGO clustering as in (I) for cell line experiments with CHIKV. Mean ± SD is shown. See also Figure S4

(I) SCI and general clustering index (GCI, binding in significant peaks) for time course experiments of CVB and SINV.

(H) ASC and viral AGO binding in percentage of total binding for time course experiments of CVB and SINV.

(F and G) BVDV electroporation time course showing BVDV (plain lines) and miR-17 (dashed lines) RNA quantification normalized to miR-21 levels (F) and AGO/miRNA binding to the BVDV 3′ UTR (G).

(E) Pie charts of miR-17 and let-7 family member abundance in cellular and BVDV chimeras. For let-7, data are divided between the two peaks of a putative canonical and non-canonical interaction.

(D) miRNA-specific chimera maps for the BVDV 3′ UTR (top), broken down by family member for miR-17 (middle) or let-7 (bottom). Canonical miRNA binding sites are highlighted in colored bars. The predicted RNA structure of interactions is shown below. Significant CIMS sites are shaded in gray. Chimera-specific CIMS sites are boxed in green (miR-17) or purple (let-7).

(A) Schematics of AGO sequestration as a function of percentage viral AGO binding in scenarios of different viral RNA levels. Definitions of AGO sequestration coefficient (ASC) and specific clustering index (SCI) are given; v, viral; t, total.

We next characterized AGO binding landscapes for a number of medically important RNA viruses and for hepatitis B virus (HBV). For both cytopathic (cp) and non-cytopathic (ncp) BVDV biotypes (differing in their natural acquisition of integrated host RNA by recombination), HBV, poliovirus (PV), coxsackie virus B3 (CVB), Sindbis virus (SINV), chikungunya virus (CHIKV), and Venezuelan equine encephalitis virus (VEEV), the read depth from AGO-viral RNA interactions was sufficient for characterization of miRNA chimeras ( Figures 2 S2 , and S3 , and Tables S1 and S2 ). The lower AGO-viral RNA read depth for yellow fever virus (YFV), dengue virus (DENV), hepatitis A virus (HAV), hepatitis E virus (HEV), influenza A virus (IAV), vesicular stomatitis virus (VSV), human metapneumovirus (hMPV), and respiratory syncytial virus (RSV) only allowed mapping of AGO binding ( Figure 3 Table S1 ).

Standard AGO maps are shown for YFV and DENVin Huh-7.5 cells (A), HAV in Huh-7.5 cells (B), HEV in Huh-7.S10.3 cells (C), VSVin A549 cells (D), IAV in A549 cells (E), and hMPVand RSVin A549 cells (F). Note the different scales for AGO binding maps. CIMS, crosslink-induced mutation sites provide nucleotide resolution for AGO-binding. Genome schematics are shown below with GFP in green and FMDV-2A in red. See also Table S1

Standard AGO (top) and miRNA-specific chimera-derived (bottom) binding maps are shown for BVDV in MDBK cells (A), HBV in Huh-7.5 cells (B), Polioin Huh-7.5 cells and CVBin HeLa cells (C), and SINV in Huh-7.5, CHIKV in Huh-7 cells, and VEEVin Huh-7.5 cells (D). Note the different y scales for AGO binding maps. Vertical zoom (insert) highlights less abundant peaks for BVDV and SINV. miRNA chimera peaks overlapping conserved canonical seed sites are indicated with boxed names ( Figure S2 ). Pie charts show miRNA abundance across all chimeras. CIMS, crosslink-induced mutation sites provide nucleotide resolution for AGO binding. Horizontal zoom is shown for BVDV and HBV. Mean of n replicates (gray) ± SEM is shown. miRNA binding sites are highlighted in colored bars. Genome schematics are shown below with GFP in green and cellular Jiv90 in cyan. Stars indicate significant peaks (p < 0.05); red stars indicate the tallest peak. See also Figures S2 and S3 and Tables S1 and S2

To specifically enrich for chimeras associated with known AGO peaks, we developed “target-CLIP.” Here, chimera enrichment is similar to CLEAR-CLIP but a set of nested primers ensures enrichment of a specific target ( Figure 1 A). Target-CLIP confirmed miR-122 binding to the HCV 5′ UTR ( Figure 1 C). These analyses validated our approach as a robust way to identify specific virus-miRNA interactions.

As proof of principle, we set out to experimentally verify HCV-miRNA interactions. To this end, we combined our previous approaches, generating viral miRNA binding landscapes using AGO-CLIP () and unambiguously identifying miRNA-target interactions () ( Figure 1 A). Analyzing miRNA chimeras from standard AGO-CLIP or CLEAR-CLIP experiments on HCV-infected Huh-7.5 hepatoma cells, we confirmed that miR-122 constitutes the vast majority of miRNA binding on the HCV 5′ UTR and at sites in the NS5B region and in the 3′ UTR ( Figures 1 B and 1C, and Tables S1 and S2 ). A minority of overall binding was attributed to other miRNAs, including miR-181-5p and miR-320a peaks in the envelope region ( Figure S1 A). We did not observe any of the non-miR-122 miRNAs previously suggested to interact with HCV. For HCVm15, where the miR-122 seed sites were substituted for miR-15 (), the 5′ UTR was bound by miR-15b and related family member miR-16 ( Figure 1 D, and Tables S1 and S2 ). For HCV-U3, where a single large cellular U3-snoRNA-derived stem loop replaces stem loop 1 and seed site 1 (), our analysis confirmed miR-122 binding at seed site 2 ( Figure 1 E, and Tables S1 and S2 ), although viral growth is largely independent of miR-122 ( Figure S1 B;). Specific miRNA binding at individual seed sites was discernable using viable recombinants harboring a site each for miR-15 and miR-122 in both configurations ( Figures 1 F and S1 C).

(D–F) miRNA binding maps as in (C) for HCVm15 (D), HCV-U3 (E), and HCVm15/122 and HCVm122/15 (F). Schematics of the HCV 5′ end with miRNA seed sites indicated are depicted in (C)–(F). See also Figure S1 and Tables S1 and S2

(C) Horizontal zoom of (B) to the HCV 5′ end. Mean of n replicates (gray) ± SEM (black lines) is shown. miRNA binding sites are highlighted in shaded bars. The miRNA-specific chimera-derived binding map and abundance pie chart is shown (middle). Target-CLIP using indicated reverse primers is shown (bottom).

(B) Standard AGO (top) and miRNA-specific chimera-derived (bottom) binding map on HCV. Vertical zoom (insert) highlights less abundant peaks. Pie chart shows miRNA abundance across all chimeras. See color legend below. CIMS, crosslink-induced mutation sites provide nucleotide resolution for AGO-binding. ∗ : significant peak, p < 0.05.

(A) Schematic of methods. Covalent ligation of miRNAs to their cognate targets creates chimeras. These are enriched in CLEAR-CLIP and target-CLIP. A further enrichment for targets of interest is achieved in target-CLIP, where RT-PCR reverse primers are designed inside AGO binding peaks identified by standard CLIP.

MicroRNA-122 antagonism against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR.

Discussion

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et al. Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs. Despite broad interest in HCV dependence on miR-122, little work has been done to look for similar interactions among other viruses. Here we employed a powerful combination of AGO-CLIP and miRNA chimera analysis to map miRNA interactions across 15 different viruses and discovered critical miR-17 and let-7 interactions for pestiviruses. Similarly to the matched tissue tropism of HCV and the liver-specific miR-122 (), the broad tropism of pestiviruses () correlates well with the distribution of miR-17 and let-7. For other viruses, significant challenges remain for assessing the functional relevance of non-specific AGO coating and interactions lacking specific miRNA chimeras. Identification of miRNA-dependent interactions could be enhanced by improvements of chimera methods. In addition, AGO-CLIP in cells devoid of mature miRNAs () could potentially identify miRNA-independent binding. Such experiments may help clarify the functional relevance, if any, of broad AGO coating for highly abundant RNA viruses such as alphaviruses. Sequestration of AGO to reduce activity of the small RNA pathway could be a parallel to subgenomic flavivirus RNA (sfRNA)-mediated inhibition of Dicer (). Further explorations of timing, cell type, and miRNA chimeras are likely to provide additional insights. Whereas essential miRNA interactions will be observed in any infection context, regulatory or inhibitory interactions, such as that of EEEV (), could be missed in certain cell subsets and in cell lines devoid of mature miRNAs ().

Luna et al., 2015 Luna J.M.

Scheel T.K.

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et al. Hepatitis C virus RNA functionally sequesters miR-122. Salmena et al., 2011 Salmena L.

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Pandolfi P.P. A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language?. In our previous studies on HCV, we demonstrated that an RNA virus can regulate hundreds of cellular transcripts through sequestration of a specific miRNA (), analogous to the concept of competing endogenous RNAs (ceRNAs) (). However, whereas most cellular miRNA targets are degraded or repressed upon miRNA binding, viral RNA replication is stimulated, acting as a positive feedback loop for additional miRNA sequestration. Here, we expanded this finding to show that pestiviruses sequester ∼40% of the miR-17 pool to de-repress cellular genes. Given the absence of pro-viral effects of the hepaci- and pestivirus-induced miRNA sponge effects, it is yet unclear whether such sponge effects are byproducts of the direct viral miRNAs dependence, or important mechanisms for manipulating host gene expression.

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et al. Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Mendell, 2008 Mendell J.T. miRiad roles for the miR-17-92 cluster in development and disease. Chase, 2013 Chase C.C. The impact of BVDV infection on adaptive immunity. Chase, 2013 Chase C.C. The impact of BVDV infection on adaptive immunity. Previously, miR-17 has been implicated in both positive and negative regulation of viral infection. Exogenous miR-17 levels were found to have a pro-viral effect for herpes simplex virus (HSV) and IAV, possibly through repression of a set of interferon-stimulated genes (). Likewise, Epstein-Barr virus (EBV) appears to co-repress cellular miR-17 targets (). In contrast, other studies showed that human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) sequester or downregulate the miR-17∼92 cluster to counteract antiviral effects (). In the immune system, overexpression of the miR-17∼92 cluster leads to lymphoproliferative disease and autoimmunity, whereas inhibition can lead to apoptosis (). Pestiviruses primarily replicate in proliferating lymphocytes (), which aligns well with elevated levels of miR-17 during proliferation. It is therefore conceivable that pestivirus-mediated sequestration of miR-17 plays a role in immunopathological events, e.g., by increasing lymphocyte apoptosis or decreasing proliferation to limit the immune response. In this context, it is noteworthy that pestiviruses induce mild to severe lymphopenia ().

Henke et al., 2008 Henke J.I.

Goergen D.

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Niepmann M. microRNA-122 stimulates translation of hepatitis C virus RNA. Sedano and Sarnow, 2014 Sedano C.D.

Sarnow P. Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2. Shimakami et al., 2012 Shimakami T.

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Lemon S.M. miR-122 stimulates hepatitis C virus RNA synthesis by altering the balance of viral RNAs engaged in replication versus translation. Mechanistic studies showed that let-7 and miR-17 have small positive effects on BVDV translation and that miR-17 exerts a small stabilizing effect on viral RNA. Similar observations for HCV were explained by the proximity and putative engagement of AGO/miR-122 with the translation initiation complex (). Concurrently, the proximity of AGO/miR-122 to the uncapped HCV RNA 5′ end may protect viral RNA from the exonucleases Xrn1 and Xrn2 (). A miR-17-dependent regulation of BVDV translation could be envisioned through genome circularization. Putative protection from exonucleases could most easily be envisioned for the 3′ exosome since miR-17 binds to the BVDV 3′ UTR. For classical flaviviruses, stable structures in the 3′ UTR do arrest Xrn1 activity to generate sfRNAs, and binding of miRNAs to the pestivirus 3′ UTR could serve a similar function, although sfRNAs have not been identified for BVDV (). Perhaps more intriguing, miRNA binding could serve as a switch between translation and replication and/or packaging. A recent study suggested that miR-122 competes with binding of cellular factors such as PCBP2 to the HCV 5′ UTR to act as such a switch (). Future work should also expand on the role, if any, of non-canonical interaction with let-7. Such an interaction could potentially protect the pestiviral 3′ end in a manner similar to how miR-122 protects the HCV 5′ end ( Figure 4 D).

In conclusion, this study outlines an effective framework for discovering virus-miRNA interactions. We determined that the HCV/miR-122 interaction is not unique, as pestiviruses critically rely on the host miR-17 and let-7 families. Antiviral therapy using miRNA antagonists may therefore also find use beyond that of HCV. Interestingly, the concept of RNA virus-induced miRNA sponging could be extended to pestiviruses in primary cell settings where sponge effects may influence the course of disease. This emerging concept in virus-host interactions holds alluring promise for future exploration for these and other viruses.