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Cell Host & Microbe

Minireview Is RNA Interference a Physiologically Relevant Innate Antiviral Immune Response in Mammals? Bryan R. Cullen,1,* Sara Cherry,2 and Benjamin R. tenOever3 1Department

of Molecular Genetics & Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, USA of Microbiology, Penn Genome Frontiers Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA 3Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2013.09.011 2Department

While RNA interference (RNAi) functions as an antiviral response in plants, nematodes, and arthropods, a similar antiviral role in mammals has remained controversial. Three recent papers provide evidence that either favors or challenges this hypothesis. Here, we discuss these new findings in the context of previous research. RNA interference (RNAi) was first described in nematodes, where injection of long double-stranded RNA (dsRNA) results in the selective destruction of cognate mRNAs (Fire et al., 1998). Subsequent work demonstrated that RNAi requires the cleavage of these long dsRNAs into 22 bp dsRNAs bearing 2 nt 30 overhangs by the RNase III enzyme Dicer. One strand of this small interfering RNA (siRNA) duplex is incorporated into the RNA-induced silencing complex (RISC) and then guides RISC to complementary mRNA species to initiate their cleavage and hydrolysis or, in an alternative mode, their translational repression (Bartel, 2009). The key component of RISC is an Argonaute (Ago) protein, 27 of which are encoded in C. elegans, although mammals only encode four Ago proteins. While RNAi can downregulate the expression of cellular mRNA species, it is likely that RNAi first evolved as an intrinsic immune response to viral infection. In particular, all RNA viruses, except retroviruses, must generate genomic complements through a virus-encoded RNA-dependent RNA polymerase, followed by transcription of the resultant complementary RNA. This yields perfect dsRNAs that can function as templates for Dicer cleavage and siRNA generation. Furthermore, viruses also produce structured RNAs and bidirectional RNAs, which can serve as Dicer substrates. The resultant siRNAs can directly target viral RNA species, and RNAi has thus been proposed as a key antiviral intrinsic immune response in plants, nematodes, and arthropods (Figure 1). In support of RNAi being a relevant antiviral mechanism, many viruses that infect these organisms have evolved viral suppressors of RNAi (VSRs) that enhance virus replication by attenuating RNAi (Wu et al., 2010). Among the best-studied VSRs are the B2 proteins encoded by members of the Nodaviridae (Aliyari et al., 2008), a family of RNA viruses that primarily infects insects but includes members that can be transmitted to vertebrates. B2 is a dsRNA binding protein that can bind both dsRNA substrates, to prevent Dicer cleavage, and siRNA duplex intermediates, to prevent RISC loading (Chao et al., 2005). While RNAi plays an important role in antiviral defense in plants and many invertebrates, evidence in favor of an analogous function for RNAi in chordates has been lacking. Deep sequencing of small RNA populations in various mammalian somatic cell lines infected by a wide range of virus species has either entirely 374 Cell Host & Microbe 14, October 16, 2013 ª2013 Elsevier Inc.

failed to detect siRNAs derived from viral RNAs or has detected these at only vanishingly low levels (e.g., Parameswaran et al., 2010). This stands in contrast with the situation in nonchordates, where siRNAs of viral origin are generally produced in readily detectable amounts (Ding and Voinnet, 2007). Similarly, while transfected long dsRNAs give rise to functional siRNAs in insect cells, transfection of dsRNAs into mammalian somatic cells does not produce functional siRNAs (Paddison et al., 2002). However, dsRNAs introduced into mammalian somatic cells do function as potent activators of the interferon response, a protein-based antiviral system that is lacking in many organisms that use RNAi as an antiviral pathway. Induction of the interferon system results in a global shutdown of mRNA translation, degradation of mRNAs, and eventually cell death (Figure 1). Interestingly, this cellular response to dsRNA also results in the posttranslational modification and inactivation of the key Ago component of RISC in mammalian cells, as demonstrated by Seo et al. (2013) in this issue of Cell Host & Microbe. While mammalian cells are able to perform RNAi when transfected with synthetic short RNA duplexes, this activity reflects the fact that a second small RNA pathway is also present (Figure 1). Colloquially referred to as siRNAs, these 22 bp duplex RNAs are in fact analogs of the microRNA (miRNA) duplex intermediates expressed by most eukaryotes. miRNAs are initially transcribed as long, largely single-stranded primary miRNA precursors that are processed by the RNA-specific endoribonuclease Drosha in the nucleus, and Dicer, in the cytoplasm, to release an 22 bp RNA duplex, bearing 2 nt 30 overhangs, that is essentially identical in structure to the siRNA duplexes released by Dicer cleavage of long dsRNA in invertebrates. As in the case of the siRNA, one strand of the miRNA duplex intermediate is loaded into RISC where it serves as a guide RNA to target RISC to complementary mRNAs. Importantly, RISCs that encounter mRNAs that are fully complementary to the miRNA can still be cleaved and degraded, so that miRNAs and synthetic siRNAs are in fact functionally indistinguishable in mammalian somatic cells (Zeng et al., 2003). While mammalian somatic cells appear incapable of effectively processing long dsRNAs into functional siRNAs, there is evidence that this is not true for mammalian germ cells and embryonic stem cells (ESCs), as well as some embryonic

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Minireview Figure 1. Cartoon Depiction of Antiviral and MicroRNA Pathways in Eukaryotes Gray bars denote the type I interferon (IFN), microRNA (miRNA), and RNA interference (RNAi) systems. Black bars above the systems denote their relative utilization as an antiviral mechanism in different species. Plants, arthropods, nematodes, and mammalian embryonic stem cells (ESCs) use both the miRNA and RNAi systems but lack a functional IFN-I system. In contrast, mammalian somatic cells have a functioning IFN-I and miRNA system, but their utilization of RNAi as an antiviral response is uncertain (depicted by the gradient of the corresponding black bar). The IFN-I system is initiated by detection of doublestranded RNA (dsRNA) by pattern recognition receptors (PRRs) resulting in the activation of many transcription factors (TFs) that induce both IFN-I and many antiviral genes that establish a protein-mediated antiviral state. The miRNA system processes dsRNA hairpins produced by the host cell that are recognized and cleaved by Dicer. One strand of the resulting duplex RNA is subsequently loaded into the RNA-induced silencing complex (RISC) where it downregulates complementary target mRNAs (blue). The RNAi system depicts viral dsRNA being cleaved by Dicer to generate small interfering RNAs (siRNAs) that are loaded into RISC to mediate an RNAdependent antiviral state by binding and cleaving viral RNAs (red).

carcinoma and teratocarcinoma cell lines (Billy et al., 2001; Paddison et al., 2002). In each of these cell types, long dsRNAs can induce the specific inhibition of cognate mRNA expression. Moreover, deep sequencing of small RNAs expressed in ESCs has identified a range of siRNAs that appear to derive from dsRNAs of endogenous origin (Tam et al., 2008). These include dsRNA derived from mobile genetic elements, such as retrotransposons, whose repression in germ cells and ESCs is clearly important to maintain genetic integrity. Of particular interest is a report showing that DNA encoding a long dsRNA complementary to the mouse c-mos gene, when introduced into the mouse genome, gave rise to readily detectable siRNA-mediated repression of c-Mos expression in mouse oocytes yet failed to generate detectable siRNAs in somatic tissues from the same mouse (Nejepinska et al., 2012). These data suggest that germ cells, ESCs, and potentially other types of pluripotent stem cells process long dsRNAs into functional siRNAs while somatic cells lack this ability. Of related interest, germ cells and ESCs also appear to be unable to mount a detectable interferon response (Burke et al., 1978), thus suggesting that RNAi and the interferon response play complementary roles in some contexts. Antiviral RNAi Responses in Embryonic Stem Cells The above data documenting the ability of ESCs to process long dsRNAs into functional siRNAs suggested that ESCs might be able to mount an RNAi response upon viral infection (Figure 1). This hypothesis was recently tested by Maillard et al. (2013) by infecting mouse ESCs with the picornavirus encephalomyocarditis virus (EMCV). EMCV-derived siRNAs of the characteristic 22 nt size were observed both by deep sequencing and north-

ern blot analysis and were derived in nearly equal amounts from both the plus and minus strands of this (+)-sense RNA virus. Virus-derived small RNAs displayed a phased 22 nt register that is characteristic of the processive Dicer cleavage of long dsRNAs. Finally, these 22 nt long EMCV-derived viral RNAs were lost in Dicer-negative ESCs, confirming their origin as Dicer cleavage products of viral RNAs. Maillard et al. (2013) next infected ESCs with a nodavirus, nodamura virus (NoV), which as noted above encodes a B2 protein, a potent VSR that functions by binding dsRNAs (Aliyari et al., 2008). Very few virus-derived siRNAs were observed upon infection of ESCs with WT NoV, but infection with a B2deficient NoV mutant (NoVDB2) produced increased levels of siRNAs as monitored by deep sequencing, even though the NoVDB2 mutant replicated at much lower levels. Importantly, analysis of NoVDB2 replication in ESCs lacking all four mammalian Ago proteins, and hence lacking functional RISCs, showed that NoVDB2 could be partially rescued, consistent with the hypothesis that the key function of B2 in NoV-infected ESCs is repression of Dicer-mediated cleavage of viral dsRNAs to generate functional antiviral siRNAs. It should be noted, however, that this same study also showed that loss of Dicer function, and hence siRNA production, in EMCV-infected ESCs did not enhance virus replication. The idea that ESCs are distinct from somatic cells was further supported by the finding that differentiation of ESCs into embryoid bodies, with loss of pluripotency markers and acquisition of tissue-specific markers, resulted in a substantial decrease in the level of EMCV-derived siRNAs, although low levels were still observed. Whether this low level of virus-derived Cell Host & Microbe 14, October 16, 2013 ª2013 Elsevier Inc. 375

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Minireview siRNA was due to a low, residual level of a factor normally missing in somatic cells was not tested, and the molecular basis for this apparent loss of RNAi competence is not currently known. Together, these data provide evidence in favor of the hypothesis that ESCs, and potentially other undifferentiated cell types with stem cell and/or pluripotent characteristics, are capable of generating siRNAs. However, it should be noted that this study did not directly demonstrate antiviral RNAi. This concept was suggested by the appearance of small RNAs and the partial rescue of NoVDB2 in the absence of functional RISC, but viral RNA cleavage or silencing was not assessed. Antiviral RNAi Responses in Somatic Cells in Culture While the evidence discussed above supporting the capacity of ESCs to generate siRNAs after viral infection is compelling, the issue of whether mammalian somatic cells can also do so remains controversial. Despite a significant amount of data suggesting the lack of an RNAi response to viral infection in chordates, Li et al. (2013) argue that somatic cells do indeed have this activity. In particular, although infection of baby hamster kidney (BHK) cells by wild-type NoV did not produce detectable viral siRNAs, infection by the NoVDB2 VSR mutant did result in small viral RNAs with the characteristic 22 nt size. Many of these small RNAs were derived from the viral () strand, which is present at low levels in cells infected by the (+)-stranded NoV. However, these reads were present at extremely low levels, just hundreds out of the millions of small RNA reads analyzed. To address whether this low level of NoV-specific siRNAs was nevertheless functionally relevant, Li et al. (2013) demonstrated that replication of NoVDB2 in BHK cells was rescued not only by expression of B2 but also by expression of the Ebola virion protein 35 (VP35), which has been reported to have VSR activity (Haasnoot et al., 2007). Of note, VP35 is in fact a dsRNA binding protein that has been shown to also block the interferon response (Prins et al., 2010). Whether NoV B2 has the ability to block the interferon response is unclear. However, dsRNAs are potent interferon inducers, and viral dsRNA binding proteins such as Ebola virus VP35 and influenza virus NS1 can antagonize this activity (Garcı´a-Sastre et al., 1998; Prins et al., 2010). Li et al. (2013) found that a NoV mutant bearing a single point mutation in B2, Arg to Glu at position 59, that blocks B2 VSR function was also highly attenuated. However, as this same mutation also blocks dsRNA binding (Aliyari et al., 2008), it does not distinguish between these distinct but related activities. As noted by Maillard et al. (2013), ‘‘demonstration of antiviral activity of siRNAs entails the genetic rescue of VSR-deficient viruses in RNAi-compromised host cells.’’ Maillard et al. (2013) demonstrated this for NoV replication in ESCs (which lack any interferon response) by showing that NoVDB2 could replicate to higher levels in ESCs lacking all mammalian Ago proteins. A similar experiment in somatic cells, for example, examining the replication capacity of NoVDB2 in mouse embryo fibroblasts lacking Dicer, would represent a critical first step to support the claim that somatic cells have the capacity to elicit an antiviral RNAi response. However, the demonstration that these viral siRNAs are indeed able to effectively silence viral mRNAs would be needed to directly prove that this pathway is indeed important. 376 Cell Host & Microbe 14, October 16, 2013 ª2013 Elsevier Inc.

To explain earlier negative data, Li et al. (2013) propose that the lack of detectable siRNAs of viral origin in somatic cells might be because mammalian viruses encode VSRs of such potency that siRNAs are not produced. They indeed observed that wild-type NoV infection of BHK cells did not produce detectable NoV siRNAs, while infection with NoVDB2 produced higher levels, albeit at very low levels. Numerous groups have shown in mammalian cells that replication of a wide range of viruses can be effectively disrupted by expression of complementary siRNAs (DeVincenzo, 2012), and viruses can be engineered to express siRNAs that can inhibit endogenous mRNAs (tenOever, 2013). Thus, RNAi and silencing pathways are active at least early after virus infection. Moreover, if all pathogenic viruses indeed express powerful VSRs, then it is difficult to explain the results of Maillard et al. showing that the picornavirus EMCV produces readily detectable levels of siRNAs in infected ESCs (Maillard et al., 2013), even though previous analysis of picornavirus-infected somatic cells in culture failed to detect analogous siRNAs (Parameswaran et al., 2010). Instead, this discrepancy suggests that there is an intrinsic difference in the ability of ESCs to generate viral siRNAs, as compared to somatic cells, that is not due to a viral activity. This idea is reinforced by data from Maillard et al. (2013) showing that differentiation of ESCs dramatically reduces their ability to produce EMCV-derived siRNAs. Furthermore, although a small number of viral dsRNA binding proteins, such as NoV B2 and Ebola virus VP35, can indeed function as VSRs, efforts to demonstrate VSR function of other viral proteins have been less successful, with recent data arguing that some previously proposed VSRs, including HIV-1 Tat and influenza virus NS1, do not block RNAi (Perez et al., 2009; Sanghvi and Steel, 2011). Indeed, the demonstration that even bacterial dsRNA binding proteins can function as repressors of RNAi when introduced into plant cells (Lichner et al., 2003) raises the question of the physiological relevance of VSR activity detected by overexpression in heterologous systems. In this context, it is particularly noteworthy that an influenza virus lacking its putative ‘‘VSR’’ maintains its lethality in interferon defective mice, suggesting that RNAi responses, if present, are insufficient to suppress replication in the context of a physiological infection (Garcı´a-Sastre et al., 1998). Lastly, Seo et al. (2013) report in this issue that viral infection leads to the inhibition of RISC function by inducing the posttranslational modification of the Ago component of RISC. These authors hypothesize that miRNA-programmed RISCs normally repress the expression of host genes involved in antiviral innate immune responses and that inhibition of RISC therefore enhances this protective response. Should RNAi represent a bona fide antiviral defense in somatic cells, it is difficult to explain how these activities could coexist. Thus, while the functional relevance of viral siRNAs in mammalian somatic cells remains an open question, it is also clear that low levels of small RNAs that resemble siRNAs are produced in some contexts. Mammalian Dicer is an exonuclease that can cleave both dsRNAs and RNA stem loops, such as pre-miRNAs. During RNA virus replication, there are undoubtedly RNAs of both types produced that could be targeted by Dicer. A key question, which remains to be addressed, is whether these small RNAs direct silencing of viral RNAs and can truly attenuate virus replication in somatic cells.

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Minireview Antiviral RNAi Responses In Vivo An important result presented by Li et al. (2013) is that infection of suckling mice by NoVDB2 leads to viral siRNAs detectable not only by deep sequencing but also by northern blot analysis. This result shows that, in contrast to cultured somatic cells, a significant number of cells in vivo that are susceptible to NoV infection are also capable of generating these RNA species. This may imply that there are significant numbers of cells with stem-cell like characteristics in vivo that maintain the ability to generate viral siRNAs. This may be particularly relevant in newborn animals, such as suckling mice, which could retain a higher number of cells with pluripotent potential than adult animals. Alternatively, it is possible that there are populations of somatic cells in vivo that retain the ability to generate siRNAs from dsRNA substrates, thus suggesting that cells cultured in vitro might not necessarily be representative of the terminally differentiated, nondividing somatic cells that predominate in vivo. Therefore, a detailed analysis of which cell types produce these viral small RNAs is essential. Li et al. (2013) demonstrate that NoVDB2 and an NoV bearing the inactivating B2 point mutation described above are both highly attenuated in vivo and differ from wild-type NoV in that they produce readily detectable viral small RNAs. However, whether the increased pathogenicity of the wild-type virus is due to the lack of viral siRNAs remains an open question. To address this question, mouse mutants lacking the ability to mount antiviral interferon responses would need to be challenged with either viral mutant. The present data show that the ability of these NoV mutants to express a functional viral dsRNA binding protein is critical to their pathogenesis, rather than definitively establishing that siRNAs are antiviral and these proteins act through antagonism of such siRNAs. Nevertheless, this result is certainly provocative and should prompt experiments designed to examine the ability of pathogenic viruses, either wild-type or mutant, to generate siRNAs in vivo and to test whether these direct viral silencing and are thus functionally relevant. Conclusion The recent publications by Maillard et al. (2013) and Li et al. (2013) have resurrected a question that seemed to have been resolved, i.e., does RNAi function as a protective antiviral immune response in mammalian cells? The data from Maillard et al. (2013) strongly suggest that ESCs are indeed able to generate siRNAs that may function to reduce viral replication. This fits nicely with the fact that the germline has additional RNA-mediated silencing pathways that protect genome integrity (Malone and Hannon, 2009). The data from Li et al. (2013) also show that mice infected with mutant forms of NoV lacking a functional B2 protein generate readily detectable levels of viral small RNAs in vivo, and these same NoV mutants can also generate siRNAs, albeit at low levels, in infected somatic cells in culture. Unfortunately, the evidence for functional relevance of these NoV-specific siRNAs in vivo or in vitro rests entirely on the hypothesis that the NoV B2 protein acts as a highly selective inhibitor of siRNA production and that the detected small RNAs are capable of eliciting antiviral RNAi. As discussed above, this is not entirely clear and would have been more convincing if the replication of these B2-deficient NoV mutants had been

shown to be rescued in RNAi-deficient cells in culture but not by mutations that inactivate dsRNA-induced interferon responses in mice. Lastly, recent data by Seo et al. (2013) suggest that RISC is in fact nonfunctional as early as 8 hr after viral infection in vivo, an observation that is difficult to reconcile with the hypothesis that RNAi functions as an important antiviral response. While the preponderance of available evidence indicates that RNAi is not a significant antiviral response in mammalian somatic cells in culture, these recent data do suggest that RNAi is active in ESCs and potentially other types of undifferentiated cells. Moreover, the evidence for high levels of siRNAs in suckling mice infected with a B2-deficient strain of NoV is consistent with the idea that siRNAs may play a protective role in vivo. Whether virus-directed RNA silencing is important in the case of human viral pathogens is a critical question that remains to be fully addressed. REFERENCES Aliyari, R., Wu, Q., Li, H.W., Wang, X.H., Li, F., Green, L.D., Han, C.S., Li, W.X., and Ding, S.W. (2008). Mechanism of induction and suppression of antiviral immunity directed by virus-derived small RNAs in Drosophila. Cell Host Microbe 4, 387–397. Bartel, D.P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233. Billy, E., Brondani, V., Zhang, H., Mu¨ller, U., and Filipowicz, W. (2001). Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc. Natl. Acad. Sci. USA 98, 14428–14433. Burke, D.C., Graham, C.F., and Lehman, J.M. (1978). Appearance of interferon inducibility and sensitivity during differentiation of murine teratocarcinoma cells in vitro. Cell 13, 243–248. Chao, J.A., Lee, J.H., Chapados, B.R., Debler, E.W., Schneemann, A., and Williamson, J.R. (2005). Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat. Struct. Mol. Biol. 12, 952–957. DeVincenzo, J.P. (2012). The promise, pitfalls and progress of RNA-interference-based antiviral therapy for respiratory viruses. Antivir. Ther. (Lond.) 17(1 Pt B), 213–225. Ding, S.W., and Voinnet, O. (2007). Antiviral immunity directed by small RNAs. Cell 130, 413–426. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811. Garcı´a-Sastre, A., Egorov, A., Matassov, D., Brandt, S., Levy, D.E., Durbin, J.E., Palese, P., and Muster, T. (1998). Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252, 324–330. Haasnoot, J., de Vries, W., Geutjes, E.J., Prins, M., de Haan, P., and Berkhout, B. (2007). The Ebola virus VP35 protein is a suppressor of RNA silencing. PLoS Pathog. 3, e86. Li, Y., Lu, J., Han, Y., Fan, X., and Ding, S.W. (2013). RNA interference functions as an antiviral immunity mechanism in mammals. Science 342, 231–234. Lichner, Z., Silhavy, D., and Burgya´n, J. (2003). Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences. J. Gen. Virol. 84, 975–980. Maillard, P.V., Ciaudo, C., Marchais, A., Li, Y., Jay, F., Ding, S.W., and Voinnet, O. (2013). Antiviral RNA interference in mammalian cells. Science 342, 235–238. Malone, C.D., and Hannon, G.J. (2009). Small RNAs as guardians of the genome. Cell 136, 656–668.

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