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RNAi-mediated antiviral immunity in mammals
Available online at www.sciencedirect.com
ScienceDirect RNAi-mediated antiviral immunity in mammals Ben Berkhout RNA interference (RNAi) was discovered in plants where it functions as the main antiviral pathway and this antiviral role was subsequently extended to invertebrates. But it remained hotly debated whether RNAi fulfils a similar role in mammals that already have a potent innate immune system based on interferon and an elaborate adaptive immune system. On the one hand, mammalian cells do encode most of the RNAi machinery, but this could be used exclusively to control cellular gene expression via micro RNAs (miRNAs). But on the other hand, virus-derived small interfering RNAs, the hallmark of RNAi involvement, could not be readily detected upon virus infection of mammalian cells. However, recent studies have indicated that these signature molecules are generated in virusinfected embryonic cell types of mammals and that viruses actively suppress such responses by means of potent RNAi suppressor proteins. Thus, the tide seems to be changing in favor of RNAi as accessory antiviral defense mechanism in humans. Intriguingly, recent studies indicate that insects have also developed an additional innate immune system that collaborates with the RNAi response in the fight against invading viral pathogens. Thus, the presence of multiple antiviral response mechanisms seems standard outside the plant world and we will specifically discuss the interactions between these antiviral programs. Address Laboratory of Experimental Virology, Department of Medical Microbiology, University of Amsterdam, Amsterdam, The Netherlands Corresponding author: Berkhout, Ben ([email protected])
Current Opinion in Virology 2018, 32:9–14 This review comes from a themed issue on Engineering for viral resistance Edited by Heiner Wedermeyer and Shouwei Ding
https://doi.org/10.1016/j.coviro.2018.07.008 1879-6257/ã 2018 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction: the history of RNAi as antiviral mechanism in plants and insects Invading viral pathogens can be cleared from the host by specific recognition and destruction of the infected cells, for example, by cytotoxic T cells of the adaptive immune system in humans. However, potent intracellular mechanisms have also been described to control a virus infection and to enable cell survival. The ancient RNA silencing www.sciencedirect.com
pathway was first described as antiviral defense mechanism in plants by Hamilton and Baulcombe [1], but key components of this mechanism are conserved among different kingdoms (fungi, animals, plants). In nematodes, it led to the discovery of the RNAi mechanism in 1998 by Andrew Fire and Craig Mello [2], who shared the 2006 Nobel Prize in Physiology. Over the last decades RNA silencing in plants and its animal counterpart RNAi have become intensively studied biological systems. RNA virus replication occurs via long double-stranded (ds) RNA intermediates that are recognized and cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs) of 21–23 nucleotides. These siRNAs are subsequently incorporated into the RNA-induced silencing complex (RISC), which uses the guide strand to recognize and cleave a complementary mRNA, which will be the viral RNA genome in the antiviral program. The Argonaute protein family plays a central role in RNA silencing processes as essential components of the RNA-induced silencing complex (RISC). Human cells encode eight Argonaute family members, of which Argonaute 2 is exclusively involved in targeted RNA cleavage as part of the RISC complex. There is a good deal of genetic and biochemical support for the importance of RNAi in antiviral defense in plants, characterized by the signature siRNA species [1,3]. Plants defective in post-transcriptional gene silencing are more susceptible to virus infections and most viral pathogens encode proteins with RNAi suppressor activity [4]. Likewise, RNAi is also a major antiviral defense mechanism in insects, for example, Drosophila melanogaster strains with an RNAi defect are highly susceptible to virus infection and these viruses — in turn — have evolved mechanisms to suppress RNAi [5–11]. In mammals, the RNAi machinery has become involved in the control of cellular gene expression at the posttranscriptional level via miRNAs that act in an exquisite sequence-specific manner. But does RNAi, on top of its essential role in controlling mammalian gene expression, also have an antiviral function in higher eukaryotes? This mini-review will deal with this pertinent question.
2. Arguments for and against an antiviral role of the RNAi mechanism in mammals Although the role of RNAi as antiviral defense mechanism has been well-established in plants and invertebrates [12], its action against mammalian pathogens has been hotly debated for some time. The major reason for this hesitation was the absence of a clear siRNA signature in virus-infected mammalian cells [13–15]. For instance, one of the pioneering deep sequencing studies in Current Opinion in Virology 2018, 32:9–14
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2004 was able to identify several virus-encoded miRNAs, but failed to detect a clear viral siRNA signal in cells infected by RNA viruses (hepatitis C virus, HCV; yellow fever virus, YFV), DNA viruses (cytomegalovirus, CMV; Epstein-Barr virus, EBV; and Kaposi sarcoma-associated herpes virus, KSHV), and the human immunodeficiency virus type 1 (HIV-1) as representative of the retrovirus family [16]. Slowly, but gradually the evidence grew in favor of an active RNAi role in the mammalian antiviral response. Several studies reported the generation of virus-specific siRNAs, for example, for HIV-1 and the LINE-1 retrotransposon, although the level of siRNA accumulation was low when compared to plant and insect systems [17,18]. An indirect indication that RNAi plays an antiviral role in mammals came from HIV-1 studies in human cells in which Dicer and Drosha expression was knocked out [19] and similar results were reported for the vesicular stomatitis virus (VSV) in mutant Caenorhabditis elegans [20]. In contrast, genetic ablation of Dicer or Argonaute 2, the major protein responsible for the siRNA pathway, failed to enhance virus replication in somatic cells [13,14,21]. Another indication of RNAimediated antiviral activity in mammals came from the description of strong RNAi suppressor activity for human pathogens, again with HIV-1 studies in the lead by identification of the multi-functional Tat protein as suppressor [17]. RNAi suppressor activity is mostly executed by a viral protein that are able to discriminate viral from cellular small RNAs, for example, VP35 protein in Ebola virus [22], the capsid protein of HCV [23] and NS1 protein of influenza virus [24], but viral transcripts may also encode RNAi suppressor activity as exemplified by the abundant VA RNA transcripts of Adenovirus that can saturate the RISC complex [25,26]. An additional argument against RNAi involvement in antiviral defense in mammals was based on the fact that humans already have two robust defense mechanism in place: the innate and adaptive immune systems [27]. On the other hand, the complete RNAi machinery is in place to execute such an antiviral function in mammals and this mechanism can definitively be exploited by man to generate mammalian cells that are resistant to specific viral pathogens. Viral resistance has indeed been established for several acute and persistent viral infections including HIV-1 [28–30], influenza virus and other respiratory pathogens [31,32] and other mammalian pathogens [33–35]. Subsequently, viral escape routes became apparent, which unequivocally demonstrated the sequencespecificity of the antiviral attack as usually a point mutation in the target sequence suffices for viral escape [36,37]. The viral escape options are limited when more conserved sequences are targeted [38,39] and combinatorial RNAi approaches were designed to prevent viral Current Opinion in Virology 2018, 32:9–14
escape [40–43]. Targeting a cellular co-factor that facilitates viral replication may also prevent viral escape, but one should be extra careful to avoid cellular toxicity [44–47]. A major complication in dissecting the contribution of the RNAi mechanism to antiviral defense in mammals is the fact that there is much interaction between the different antiviral programs. The NS1 protein of influenza virus may underscore this possibility as this RNA effector does also influence the innate interferon system and possibly the adaptive immune system [48–51]. In fact, most viral suppressor proteins are double-stranded RNA (dsRNA)binding proteins that could affect both the RNAi and interferon responses. A functional link between the RNAi response and interferon innate immune response pathways was suggested early on [52]. Major dsRNA sensors include the pattern recognition receptors like Toll-like receptors and retinoic-acid-inducible protein-1/melanoma-differentiation-associated gene 5 (RIG-1/MD5), a cytosolic innate immune protein complex that senses viral dsRNA with a 50 -triphosphate overhang [53,54], but also the antiviral proteins 20 –50 OAS/RNaseL/PKR [55]. Viral dsRNA molecules are thus not only recognized by Dicer, but also by RIG-I-MDA5 and Toll-like receptor 3 to induce the antiviral interferon-response against the invading virus [56]. The interferon response can also suppress the RNAi program by inhibiting RISC [57,58]. Interferon will also induce an additional layer of protection by induction of antiviral restriction factors that form an integral part of the host innate immune system. Again, pioneering HIV-1 studies indicated that human cells encode multiple restriction factors including APOBEC3G, TRIM5a, tetherin, SERINC5, SAMHD1 and ZAP that block different phases of the virus replication cycle. It is likely that many more restriction factors will be identified, especially for other viral pathogens, and some may have specialized to counter different viruses. We will focus on the cellular RNA sensors that detect virus-specific molecular signatures, but realize that the microbial world is more complex, for example, with additional control mechanisms with DNA-sensors as IFI16 and cGAS. It is worth mentioning that several unique and intricate virus–RNAi interactions have been discovered that may also have troubled a straightforward dissection of antiviral effects of the RNAi mechanism in mammals [59]. The most notable example is the dependence of HCV on the cellular miR-122 to support viral RNA translation and replication [60,61]. Cellular miRNAs can also contribute to the innate immune system against viruses, as exemplified by miR-32 that downregulates five cellular co-factors that support the replication of the primate retrovirus prototype foamy virus (PFV) in human cells [62] and cellular miRNAs that exhibit activity against HIV-1, VSV and influenza virus [63]. The interferon pathway has also www.sciencedirect.com
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been reported to trigger the expression of specific cellular miRNAs with an antiviral activity, for example, several miRNAs that are almost perfectly complementary to the HCV RNA genome [64], and the HCV-enhancing miR122 was reported to be downregulated [61]. Host miRNAs can also modulate cellular genes that are involved in the antiviral interferon response. All these complexities clearly distract from the central question: does RNAi have an antiviral role in mammalian cells that already have the interferon response as innate immune system to recognize viral dsRNA.
3. Recent evidence in favor of an antiviral function of RNAi in mammals A virus-specific siRNA signature became more apparent in two 2013 studies on virus-infected mammalian embryonic stem cells that do not have a functional interferon system [65,66]. Maillard et al used deep sequencing to reveal the accumulation of 22-nucleotide RNAs in undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus (NoV), a mosquito-transmitted RNA virus and thus not a human virus. These siRNA are processed from viral dsRNA replication intermediates into successive pieces that are typical of Dicer-mediated processing and this diagnostic hallmark of RNAi action decreased in abundance upon cell differentiation. Furthermore, a NoV mutant lacking the RNAi suppressor that antagonized Dicer is rescued in RNAi-deficient cells [66]. Li et al also reported the accumulation of viral siRNAs in cultured hamster cells and suckling mice infected by the mutant NoV that lacks the RNAi suppressor protein B2, but not wild-type NoV [65]. Importantly, the RNAi response was linked to a potent antiviral activity that made suckling mice resistant to an otherwise lethal NoV infection. These two studies demonstrated that the catalytic RNAi mechanism indeed is one of several defense layers that mammals have evolved to combat RNA virus infections and thus ended a nearly 10-year period of intense debate and controversy [67]. Subsequently, the antiviral RNAi involvement was confirmed for human cells infected by influenza virus mutants deleted for the RNAi suppressor [68] and in cells in which the interferon pathway was inactivated [57]. Similarities in the induction and suppression of the antiviral RNAi program by closely related viruses in fruit flies, nematodes and mammals led to the conclusion that this program is conserved across the animal kingdom [65]. Not unimportantly, these two 2013 studies also provided clues as to why mammalian antiviral RNAi had remained elusive up to then. First, most previous studies used virulent virus strains that encode potent RNAi suppressors that prevent the massive production of the diagnostic siRNAs. Second, the siRNA level was approximately one order of magnitude higher in mouse embryonic stem cells www.sciencedirect.com
(mESCs) than in differentiated mESCs induced in cell culture [66]. In general, undifferentiated cells also express a reduced level of interferon. These combined findings suggest an intriguing dichotomy whereby differentiated, somatic cells rely solely on the protein-based IFN response, while undifferentiated pluripotent cells can utilize RNAi (Table 1). In 2017, Qiu et al. demonstrated that the human enterovirus 71 (HEV71), the etiological agent of hand-food-andmouth disease, encodes the 3A protein as RNAi suppressor and that 3A-deficient virus mutants induce the production of viral siRNAs in somatic cells and mice [69]. Most importantly, this group also reported that these siRNAs load into Ago to trigger degradation of the cognate viral RNA genome. The virus-induced RNAi response was proposed to confer antiviral immunity as the 3A-deficient HEV71 mutant demonstrated a severe replication defect and Dicer-deficiency successfully restored virus replication. This study confirmed that the initial difficulty of detecting RNAi in virus-infected mammalian cells is due to the expression of highly effective viral RNAi suppressors. This wealth of evidence even convinced one of the most critical voices about the antiviral role of RNAi in mammals, at least in undifferentiated cells (68). To exclude an interferon-mediated effect, it was argued that one needs to test wild-type and suppressor-lacking virus mutants in systems that lack a functional interferon response [70]. The Qiu et al study fulfilled this requirement in two ways, by blocking the interferon response with a drug and by using mouse cells that lack a functional interferon receptor [69].
4. Recent evidence that invertebrates also evolved multiple antiviral pathways Recent evidence indicates that the virus-specific RNAi response in insects is amplified by a novel mechanism in which the viral RNA is first converted into linear DNA and subsequently into the more stable circular DNA form (cvDNA) [71,72,73]. Reverse transcription is executed by a cellular AZT-sensitive reverse transcriptase encoded by retrotransposons that are lacking from human cells, but important molecular details of this process are still lacking. It was speculated that cvDNA could be further ‘stabilized’ by integration into the host cell genome. Interestingly, more of these endogenized viral elements Table 1 The major antiviral pathways in plants, invertebrates and mammals Plants Invertebrates
RNAi
Mammals Undifferentiated and cultured mature cells
1. RNAi 1. RNAi 2. Innate (DVG) 2. Innate (interferon) 3. Adaptive (Ab)
Mammals Adult tissues 1. Innate (interferon) 2. Adaptive (Ab)
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have recently been described and such endogenous viral elements were linked to transposons in the mosquito genome [74]. Anyhow, the unique cvDNA product suffices to produce siRNAs that confer partial protection to Drosophila against arbovirus challenge. Interestingly, cvDNA synthesis is not regulated by the RNAi-associated dicing activity of Dicer-2, but rather by its helicase domain that mimics, both structurally and functionally, the RIG-I-like receptors (RLRs) [75,76]. These RLRs recognize defective viral genomes (DVGs) carrying truncations and rearrangements, possibly because their subcellular localization differs from the replication-competent full-length viral genomes [77]. DVGs were thus suggested to be more than just a byproduct of massive virus replication as they trigger the host immune response and thereby modulate the fate of infection [77]. These findings support the notion that RNAi is part of a multifaceted, yet interconnected network of cellular defense pathways against not only invading RNA viruses, but likely also other pathogens and potentially hazardous mobile genetic elements [78]. In analogy with the machinery that induces type 1 interferon and other immune responses in mammals, Dicer-2 was suggested to act as pattern recognition receptor (PRR) and the DVG as pathogen-associated molecular patterns (PAMPs). Thus, a convergence between vertebrate antiviral immunity and the mammalian type 1 interferon response was suggested, as indicated by the color-coding in Table 1. Convergence has also been reported for suppression by viruses of the interferon and RNAi pathways. Several viral proteins have been suggested to exhibit suppressive activity against both immune pathways, for example, the influenza virus NS1 protein [24,50] and the Ebola virus VP35 protein [22]. This mimics the prevalence of RNAi suppressor proteins by plant viruses and even some trans-kingdom activity has even been reported for a plant virus suppressor in human cells and vice versa [79,80]. Thus, it now seems that only plants rely on a single antiviral defense mechanism (RNAi), whereas invertebrates and mammals have evolved multiple systems, at least 2 and 3 mechanisms, respectively (Table 1). Mammals are unique in having evolved the adaptive arm of immunity that includes potent antibodies (Ab), but this apparently does not suffice for the control of pathogens, for example, during the acute phase of infection or in the context of non-differentiated embryonic cell.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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siRNAs that confer antiviral immunity upon loading into Ago to degrade the cognate HEV71 RNA. 70. Cullen BR, Cherry S, tenOever BR: Is RNA interference a physiologically relevant innate antiviral immune response in mammals? Cell Host Microbe 2013, 14:374-378. 71. Goic B, Vodovar N, Mondotte JA, Monot C, Frangeul L, Blanc H, Gausson V, Vera-Otarola J, Cristofari G, Saleh MC: RNAmediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila. Nat Immunol 2013, 14:396-403. 72. Goic B, Stapleford KA, Frangeul L, Doucet AJ, Gausson V, Blanc H, Schemmel-Jofre N, Cristofari G, Lambrechts L, Vignuzzi M et al.: Virus-derived DNA drives mosquito vector tolerance to arboviral infection. Nat Commun 2016, 7:12410. 73. Poirier EZ, Goic B, Tome-Poderti L, Frangeul L, Boussier J, Gausson V, Blanc H, Vallet T, Loyd H, Levi LI et al.: Dicer-2dependent generation of viral DNA from defective genomes of RNA viruses modulates antiviral immunity in Insects. Cell Host Microbe 2018, 23:353-365 e358. Broadening of the antiviral program in insects. 74. Suzuki Y, Frangeul L, Dickson LB, Blanc H, Verdier Y, Vinh J, Lambrechts L, Saleh MC: Uncovering the repertoire of endogenous flaviviral elements in Aedes mosquito genomes. J Virol 2017, 91:e00571. 75. Paro S, Imler JL, Meignin C: Sensing viral RNAs by Dicer/RIG-I like ATPases across species. Curr Opin Immunol 2015, 32:106113. 76. Baum A, Sachidanandam R, Garcia-Sastre A: Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing. Proc Natl Acad Sci U S A 2010, 107:16303-16308. 77. Xu J, Sun Y, Li Y, Ruthel G, Weiss SR, Raj A, Beiting D, Lopez CB: Replication defective viral genomes exploit a cellular prosurvival mechanism to establish paramyxovirus persistence. Nat Commun 2017, 8:799. 78. Ghildiyal M, Seitz H, Horwich MD, Li C, Du T, Lee S, Xu J, Kittler EL, Zapp ML, Weng Z et al.: Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 2008, 320:1077-1081. 79. Schnettler E, de VW, Hemmes H, Haasnoot J, Kormelink R, Goldbach R, Berkhout B: The NS3 protein of rice Hoja blanca virus complements the RNAi suppressor function of HIV-1 Tat. EMBO Rep 2009, 10:258-263. Trans-kingdom activity of the HIV-1 RNA suppressor protein Tat in plants. 80. Qian S, Zhong X, Yu L, Ding B, de Haan P, Boris-Lawrie K: HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1. Proc Natl Acad Sci U S A 2009, 106:605-610.
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ScienceDirect RNAi-mediated antiviral immunity in mammals Ben Berkhout RNA interference (RNAi) was discovered in plants where it functions as the main antiviral pathway and this antiviral role was subsequently extended to invertebrates. But it remained hotly debated whether RNAi fulfils a similar role in mammals that already have a potent innate immune system based on interferon and an elaborate adaptive immune system. On the one hand, mammalian cells do encode most of the RNAi machinery, but this could be used exclusively to control cellular gene expression via micro RNAs (miRNAs). But on the other hand, virus-derived small interfering RNAs, the hallmark of RNAi involvement, could not be readily detected upon virus infection of mammalian cells. However, recent studies have indicated that these signature molecules are generated in virusinfected embryonic cell types of mammals and that viruses actively suppress such responses by means of potent RNAi suppressor proteins. Thus, the tide seems to be changing in favor of RNAi as accessory antiviral defense mechanism in humans. Intriguingly, recent studies indicate that insects have also developed an additional innate immune system that collaborates with the RNAi response in the fight against invading viral pathogens. Thus, the presence of multiple antiviral response mechanisms seems standard outside the plant world and we will specifically discuss the interactions between these antiviral programs. Address Laboratory of Experimental Virology, Department of Medical Microbiology, University of Amsterdam, Amsterdam, The Netherlands Corresponding author: Berkhout, Ben ([email protected])
Current Opinion in Virology 2018, 32:9–14 This review comes from a themed issue on Engineering for viral resistance Edited by Heiner Wedermeyer and Shouwei Ding
https://doi.org/10.1016/j.coviro.2018.07.008 1879-6257/ã 2018 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction: the history of RNAi as antiviral mechanism in plants and insects Invading viral pathogens can be cleared from the host by specific recognition and destruction of the infected cells, for example, by cytotoxic T cells of the adaptive immune system in humans. However, potent intracellular mechanisms have also been described to control a virus infection and to enable cell survival. The ancient RNA silencing www.sciencedirect.com
pathway was first described as antiviral defense mechanism in plants by Hamilton and Baulcombe [1], but key components of this mechanism are conserved among different kingdoms (fungi, animals, plants). In nematodes, it led to the discovery of the RNAi mechanism in 1998 by Andrew Fire and Craig Mello [2], who shared the 2006 Nobel Prize in Physiology. Over the last decades RNA silencing in plants and its animal counterpart RNAi have become intensively studied biological systems. RNA virus replication occurs via long double-stranded (ds) RNA intermediates that are recognized and cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs) of 21–23 nucleotides. These siRNAs are subsequently incorporated into the RNA-induced silencing complex (RISC), which uses the guide strand to recognize and cleave a complementary mRNA, which will be the viral RNA genome in the antiviral program. The Argonaute protein family plays a central role in RNA silencing processes as essential components of the RNA-induced silencing complex (RISC). Human cells encode eight Argonaute family members, of which Argonaute 2 is exclusively involved in targeted RNA cleavage as part of the RISC complex. There is a good deal of genetic and biochemical support for the importance of RNAi in antiviral defense in plants, characterized by the signature siRNA species [1,3]. Plants defective in post-transcriptional gene silencing are more susceptible to virus infections and most viral pathogens encode proteins with RNAi suppressor activity [4]. Likewise, RNAi is also a major antiviral defense mechanism in insects, for example, Drosophila melanogaster strains with an RNAi defect are highly susceptible to virus infection and these viruses — in turn — have evolved mechanisms to suppress RNAi [5–11]. In mammals, the RNAi machinery has become involved in the control of cellular gene expression at the posttranscriptional level via miRNAs that act in an exquisite sequence-specific manner. But does RNAi, on top of its essential role in controlling mammalian gene expression, also have an antiviral function in higher eukaryotes? This mini-review will deal with this pertinent question.
2. Arguments for and against an antiviral role of the RNAi mechanism in mammals Although the role of RNAi as antiviral defense mechanism has been well-established in plants and invertebrates [12], its action against mammalian pathogens has been hotly debated for some time. The major reason for this hesitation was the absence of a clear siRNA signature in virus-infected mammalian cells [13–15]. For instance, one of the pioneering deep sequencing studies in Current Opinion in Virology 2018, 32:9–14
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2004 was able to identify several virus-encoded miRNAs, but failed to detect a clear viral siRNA signal in cells infected by RNA viruses (hepatitis C virus, HCV; yellow fever virus, YFV), DNA viruses (cytomegalovirus, CMV; Epstein-Barr virus, EBV; and Kaposi sarcoma-associated herpes virus, KSHV), and the human immunodeficiency virus type 1 (HIV-1) as representative of the retrovirus family [16]. Slowly, but gradually the evidence grew in favor of an active RNAi role in the mammalian antiviral response. Several studies reported the generation of virus-specific siRNAs, for example, for HIV-1 and the LINE-1 retrotransposon, although the level of siRNA accumulation was low when compared to plant and insect systems [17,18]. An indirect indication that RNAi plays an antiviral role in mammals came from HIV-1 studies in human cells in which Dicer and Drosha expression was knocked out [19] and similar results were reported for the vesicular stomatitis virus (VSV) in mutant Caenorhabditis elegans [20]. In contrast, genetic ablation of Dicer or Argonaute 2, the major protein responsible for the siRNA pathway, failed to enhance virus replication in somatic cells [13,14,21]. Another indication of RNAimediated antiviral activity in mammals came from the description of strong RNAi suppressor activity for human pathogens, again with HIV-1 studies in the lead by identification of the multi-functional Tat protein as suppressor [17]. RNAi suppressor activity is mostly executed by a viral protein that are able to discriminate viral from cellular small RNAs, for example, VP35 protein in Ebola virus [22], the capsid protein of HCV [23] and NS1 protein of influenza virus [24], but viral transcripts may also encode RNAi suppressor activity as exemplified by the abundant VA RNA transcripts of Adenovirus that can saturate the RISC complex [25,26]. An additional argument against RNAi involvement in antiviral defense in mammals was based on the fact that humans already have two robust defense mechanism in place: the innate and adaptive immune systems [27]. On the other hand, the complete RNAi machinery is in place to execute such an antiviral function in mammals and this mechanism can definitively be exploited by man to generate mammalian cells that are resistant to specific viral pathogens. Viral resistance has indeed been established for several acute and persistent viral infections including HIV-1 [28–30], influenza virus and other respiratory pathogens [31,32] and other mammalian pathogens [33–35]. Subsequently, viral escape routes became apparent, which unequivocally demonstrated the sequencespecificity of the antiviral attack as usually a point mutation in the target sequence suffices for viral escape [36,37]. The viral escape options are limited when more conserved sequences are targeted [38,39] and combinatorial RNAi approaches were designed to prevent viral Current Opinion in Virology 2018, 32:9–14
escape [40–43]. Targeting a cellular co-factor that facilitates viral replication may also prevent viral escape, but one should be extra careful to avoid cellular toxicity [44–47]. A major complication in dissecting the contribution of the RNAi mechanism to antiviral defense in mammals is the fact that there is much interaction between the different antiviral programs. The NS1 protein of influenza virus may underscore this possibility as this RNA effector does also influence the innate interferon system and possibly the adaptive immune system [48–51]. In fact, most viral suppressor proteins are double-stranded RNA (dsRNA)binding proteins that could affect both the RNAi and interferon responses. A functional link between the RNAi response and interferon innate immune response pathways was suggested early on [52]. Major dsRNA sensors include the pattern recognition receptors like Toll-like receptors and retinoic-acid-inducible protein-1/melanoma-differentiation-associated gene 5 (RIG-1/MD5), a cytosolic innate immune protein complex that senses viral dsRNA with a 50 -triphosphate overhang [53,54], but also the antiviral proteins 20 –50 OAS/RNaseL/PKR [55]. Viral dsRNA molecules are thus not only recognized by Dicer, but also by RIG-I-MDA5 and Toll-like receptor 3 to induce the antiviral interferon-response against the invading virus [56]. The interferon response can also suppress the RNAi program by inhibiting RISC [57,58]. Interferon will also induce an additional layer of protection by induction of antiviral restriction factors that form an integral part of the host innate immune system. Again, pioneering HIV-1 studies indicated that human cells encode multiple restriction factors including APOBEC3G, TRIM5a, tetherin, SERINC5, SAMHD1 and ZAP that block different phases of the virus replication cycle. It is likely that many more restriction factors will be identified, especially for other viral pathogens, and some may have specialized to counter different viruses. We will focus on the cellular RNA sensors that detect virus-specific molecular signatures, but realize that the microbial world is more complex, for example, with additional control mechanisms with DNA-sensors as IFI16 and cGAS. It is worth mentioning that several unique and intricate virus–RNAi interactions have been discovered that may also have troubled a straightforward dissection of antiviral effects of the RNAi mechanism in mammals [59]. The most notable example is the dependence of HCV on the cellular miR-122 to support viral RNA translation and replication [60,61]. Cellular miRNAs can also contribute to the innate immune system against viruses, as exemplified by miR-32 that downregulates five cellular co-factors that support the replication of the primate retrovirus prototype foamy virus (PFV) in human cells [62] and cellular miRNAs that exhibit activity against HIV-1, VSV and influenza virus [63]. The interferon pathway has also www.sciencedirect.com
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been reported to trigger the expression of specific cellular miRNAs with an antiviral activity, for example, several miRNAs that are almost perfectly complementary to the HCV RNA genome [64], and the HCV-enhancing miR122 was reported to be downregulated [61]. Host miRNAs can also modulate cellular genes that are involved in the antiviral interferon response. All these complexities clearly distract from the central question: does RNAi have an antiviral role in mammalian cells that already have the interferon response as innate immune system to recognize viral dsRNA.
3. Recent evidence in favor of an antiviral function of RNAi in mammals A virus-specific siRNA signature became more apparent in two 2013 studies on virus-infected mammalian embryonic stem cells that do not have a functional interferon system [65,66]. Maillard et al used deep sequencing to reveal the accumulation of 22-nucleotide RNAs in undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus (NoV), a mosquito-transmitted RNA virus and thus not a human virus. These siRNA are processed from viral dsRNA replication intermediates into successive pieces that are typical of Dicer-mediated processing and this diagnostic hallmark of RNAi action decreased in abundance upon cell differentiation. Furthermore, a NoV mutant lacking the RNAi suppressor that antagonized Dicer is rescued in RNAi-deficient cells [66]. Li et al also reported the accumulation of viral siRNAs in cultured hamster cells and suckling mice infected by the mutant NoV that lacks the RNAi suppressor protein B2, but not wild-type NoV [65]. Importantly, the RNAi response was linked to a potent antiviral activity that made suckling mice resistant to an otherwise lethal NoV infection. These two studies demonstrated that the catalytic RNAi mechanism indeed is one of several defense layers that mammals have evolved to combat RNA virus infections and thus ended a nearly 10-year period of intense debate and controversy [67]. Subsequently, the antiviral RNAi involvement was confirmed for human cells infected by influenza virus mutants deleted for the RNAi suppressor [68] and in cells in which the interferon pathway was inactivated [57]. Similarities in the induction and suppression of the antiviral RNAi program by closely related viruses in fruit flies, nematodes and mammals led to the conclusion that this program is conserved across the animal kingdom [65]. Not unimportantly, these two 2013 studies also provided clues as to why mammalian antiviral RNAi had remained elusive up to then. First, most previous studies used virulent virus strains that encode potent RNAi suppressors that prevent the massive production of the diagnostic siRNAs. Second, the siRNA level was approximately one order of magnitude higher in mouse embryonic stem cells www.sciencedirect.com
(mESCs) than in differentiated mESCs induced in cell culture [66]. In general, undifferentiated cells also express a reduced level of interferon. These combined findings suggest an intriguing dichotomy whereby differentiated, somatic cells rely solely on the protein-based IFN response, while undifferentiated pluripotent cells can utilize RNAi (Table 1). In 2017, Qiu et al. demonstrated that the human enterovirus 71 (HEV71), the etiological agent of hand-food-andmouth disease, encodes the 3A protein as RNAi suppressor and that 3A-deficient virus mutants induce the production of viral siRNAs in somatic cells and mice [69]. Most importantly, this group also reported that these siRNAs load into Ago to trigger degradation of the cognate viral RNA genome. The virus-induced RNAi response was proposed to confer antiviral immunity as the 3A-deficient HEV71 mutant demonstrated a severe replication defect and Dicer-deficiency successfully restored virus replication. This study confirmed that the initial difficulty of detecting RNAi in virus-infected mammalian cells is due to the expression of highly effective viral RNAi suppressors. This wealth of evidence even convinced one of the most critical voices about the antiviral role of RNAi in mammals, at least in undifferentiated cells (68). To exclude an interferon-mediated effect, it was argued that one needs to test wild-type and suppressor-lacking virus mutants in systems that lack a functional interferon response [70]. The Qiu et al study fulfilled this requirement in two ways, by blocking the interferon response with a drug and by using mouse cells that lack a functional interferon receptor [69].
4. Recent evidence that invertebrates also evolved multiple antiviral pathways Recent evidence indicates that the virus-specific RNAi response in insects is amplified by a novel mechanism in which the viral RNA is first converted into linear DNA and subsequently into the more stable circular DNA form (cvDNA) [71,72,73]. Reverse transcription is executed by a cellular AZT-sensitive reverse transcriptase encoded by retrotransposons that are lacking from human cells, but important molecular details of this process are still lacking. It was speculated that cvDNA could be further ‘stabilized’ by integration into the host cell genome. Interestingly, more of these endogenized viral elements Table 1 The major antiviral pathways in plants, invertebrates and mammals Plants Invertebrates
RNAi
Mammals Undifferentiated and cultured mature cells
1. RNAi 1. RNAi 2. Innate (DVG) 2. Innate (interferon) 3. Adaptive (Ab)
Mammals Adult tissues 1. Innate (interferon) 2. Adaptive (Ab)
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have recently been described and such endogenous viral elements were linked to transposons in the mosquito genome [74]. Anyhow, the unique cvDNA product suffices to produce siRNAs that confer partial protection to Drosophila against arbovirus challenge. Interestingly, cvDNA synthesis is not regulated by the RNAi-associated dicing activity of Dicer-2, but rather by its helicase domain that mimics, both structurally and functionally, the RIG-I-like receptors (RLRs) [75,76]. These RLRs recognize defective viral genomes (DVGs) carrying truncations and rearrangements, possibly because their subcellular localization differs from the replication-competent full-length viral genomes [77]. DVGs were thus suggested to be more than just a byproduct of massive virus replication as they trigger the host immune response and thereby modulate the fate of infection [77]. These findings support the notion that RNAi is part of a multifaceted, yet interconnected network of cellular defense pathways against not only invading RNA viruses, but likely also other pathogens and potentially hazardous mobile genetic elements [78]. In analogy with the machinery that induces type 1 interferon and other immune responses in mammals, Dicer-2 was suggested to act as pattern recognition receptor (PRR) and the DVG as pathogen-associated molecular patterns (PAMPs). Thus, a convergence between vertebrate antiviral immunity and the mammalian type 1 interferon response was suggested, as indicated by the color-coding in Table 1. Convergence has also been reported for suppression by viruses of the interferon and RNAi pathways. Several viral proteins have been suggested to exhibit suppressive activity against both immune pathways, for example, the influenza virus NS1 protein [24,50] and the Ebola virus VP35 protein [22]. This mimics the prevalence of RNAi suppressor proteins by plant viruses and even some trans-kingdom activity has even been reported for a plant virus suppressor in human cells and vice versa [79,80]. Thus, it now seems that only plants rely on a single antiviral defense mechanism (RNAi), whereas invertebrates and mammals have evolved multiple systems, at least 2 and 3 mechanisms, respectively (Table 1). Mammals are unique in having evolved the adaptive arm of immunity that includes potent antibodies (Ab), but this apparently does not suffice for the control of pathogens, for example, during the acute phase of infection or in the context of non-differentiated embryonic cell.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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