Advertisement
  • Loading metrics

Mammalian antiviral systems directed by small RNA

Mammalian antiviral systems directed by small RNA

  • Tomoko Takahashi, 
  • Steven M. Heaton, 
  • Nicholas F. Parrish
PLOS
x

Abstract

There are strong incentives for human populations to develop antiviral systems. Similarly, genomes that encode antiviral systems have had strong selective advantages. Protein-guided immune systems, which have been well studied in mammals, are necessary for survival in our virus-laden environments. Small RNA–directed antiviral immune systems suppress invasion of cells by non-self genetic material via complementary base pairing with target sequences. These RNA silencing-dependent systems operate in diverse organisms. In mammals, there is strong evidence that microRNAs (miRNAs) regulate endogenous genes important for antiviral immunity, and emerging evidence that virus-derived nucleic acids can be directly targeted by small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and transfer RNAs (tRNAs) for protection in some contexts. In this review, we summarize current knowledge of the antiviral functions of each of these small RNA types and consider their conceptual and mechanistic overlap with innate and adaptive protein-guided immunity, including mammalian antiviral cytokines, as well as the prokaryotic RNA-guided immune system, CRISPR. In light of recent successes in delivery of RNA for antiviral purposes, most notably for vaccination, we discuss the potential for development of small noncoding RNA–directed antiviral therapeutics and prophylactics.

Author summary

Viruses are all around us and are likely inside some of the reader’s cells at this moment. Organisms are accommodated to this reality and encode various immune systems to limit virus replication. In mammals, the best studied immune systems are directed by proteins that specifically recognize viruses. These include diverse antibodies and T cell receptors, which recognize viral proteins, and pattern recognition receptors, some of which can recognize viral nucleic acids. In other organisms, including bacteria, immune systems directed by small RNAs are also well known; spacer-derived guide RNAs in CRISPR/Cas immune systems are one prominent example. The small RNAs directing these systems derive their specificity via complementary base pairing with their targets, which include both host and viral nucleic acids. Rather than having “traded in” these systems for more advanced protein-directed systems, increasing evidence supports the perspective that small RNA–directed immune systems remain active in mammalian antiviral immunity in some contexts. Here, we review what is known so far about the emerging roles of mammalian siRNAs, miRNAs, piRNAs, and tRNAs in directing immunity to viruses.

Introduction

Viruses are the most abundant and genetically diverse entity in our biosphere and exist wherever life is found [1]. Viruses are characterized by dependence on host genome-encoded protein products and other materials (e.g., plasma membrane) for their replication, whereas host cellular and organismal replication are in principle independent of the genetic material of viruses. Replication of some viruses is associated with cellular and organismal death. Virus replication has thus often been interpreted to be detrimental to their hosts at evolutionary, organismal, and cellular scales of analysis (although host-virus antagonism is not a comprehensive picture of virus–host ecology, e.g., see [2]). Numerous systems that confer resistance to or otherwise mitigate virus infection have been identified. Immune systems capable of distinguishing “self” from “non-self” and eliminating “non-self” are inferred to have been established and refined through antagonistic interactions between viruses and their hosts over long periods of time [35]; mammalian genomes encode various complex systems known to mediate antiviral functions.

Components of mammalian immune systems are often categorized as belonging to innate or adaptive arms based on generalizably distinct functional and evolutionary features. When a virus infects human cells, innate immunity is activated first, followed by adaptive immunity. Innate immunity is activated by features common to invading pathogens, termed pathogen-associated molecular patterns (PAMPs). PAMPs are recognized by sensor proteins such as Toll-like receptors (TLRs) and RIG-I like receptors (RLRs) [69]. Upon activation, these receptors induce production of various antiviral cytokines, such as type-I interferon (IFN). Cytokines stimulate expression of antiviral genes, which repress viral replication and recruit or activate specialized cells including phagocytes and antigen-presenting cells. Adaptive immune lymphocytes generate vastly diverse receptors through gene recombination and then selectively proliferate on the basis of their receptor’s specificity. This enables high specificity as well as anamnestic properties dependent on long-lived “memory” cells. Conventional mammalian adaptive immunity is targeted to presented protein antigens and is characterized by relative evolutionary novelty; the critical genes are conserved only among jawed vertebrates, although adaptive systems have evolved convergently in other lineages [10]. Loss-of-function mutation of some immune genes has been associated with increased susceptibility to fatal virus infection before reproductive maturity [11], arguing strongly that antiviral activity is among their evolved functions [12].

Recent studies have suggested that antiviral immune systems directed by small RNAs are active in mammals (Table 1) [1318]. Like proteinaceous innate immune receptors, some RNA-based antiviral systems are broadly expressed, such as those directed by microRNAs (miRNAs), whereas others, such as those reportedly mediated by siRNAs, are active in specialized cell types. Some small RNA–directed responses are amplified in a feedforward manner reminiscent of the expansion of virus-specific lymphocyte clones in adaptive immunity. In general, however, the reported small RNA–directed immune mechanisms defy categorization as innate or adaptive; for example, some can respond immediately after viral infection, yet also have high specificity mediated by complementary base pairing with their target sequences. The evolutionary history of the RNAs guiding these systems also run the gamut from tightly constrained mature tRNAs, as ancient as cellular life [19], to piRNAs, notable for the remarkable plasticity of their targeting capacity even within a species [20]. Evidence of small RNA–directed immunity has emerged in the postgenomic era; our understanding of how the constituent components interact at scales larger than individual cells to produce organismal phenotypes is more limited than for classical immunology [21]. Functional and evolutionary categorizations are thus less useful to organize an overview of RNA-guided immunity than they are for protein-guided systems; here, we will summarize the current knowledge regarding antiviral immune functions directed by small noncoding RNAs grouped on the basis of their biogenesis. Notably, biochemical definitions of these molecules and their cofactors preceded recognition of their antiviral properties of the RNAs in each case.

Viral RNA elimination by siRNA-directed RNA interference

The best studied example of small RNA–directed immunity is that guided by viral siRNAs (Fig 1). When RNA or DNA viruses infect cells, viral origin double-stranded RNAs (dsRNAs) can be generated either as replication intermediates or as structured regions of viral genetic material. These dsRNAs can be cleaved by the endoribonuclease Dicer, which is well conserved in chordates [22], into siRNAs, which are characteristically 21 to 24 nucleotides (nt) in length with a 2-nt 3′ overhang. One of the resulting strands, the guide strand, is loaded onto an argonaute clade protein (AGO), also an endonuclease, and base pairs with complementary viral RNA. AGOs are core components of the RNA-induced silencing complex (RISC) and some trigger the cleavage of the viral RNA, resulting in the repression of viral replication. Here, we refer to this system as antiviral RNAi, and it functions as a potent defense system in plants and invertebrates [23,24]. In contrast, whether antiviral RNAi functions as a meaningful aspect of mammalian defenses against viruses is controversial. Recent reviews have cataloged the evidence in favor of this activity [25], and, since 2020, experiments with knockout mouse challenge have shown that Ago4 is involved in mouse defense against influenza virus [26] and Ago2 with defense against Nodamura virus (NoV) lacking B2 (see below) [27]. The mechanisms proposed to limit the effectiveness of antiviral siRNAs in mammals are potentially relevant to the antiviral activity of other small RNAs as well, so here we will focus on them.

thumbnail
Fig 1. Overview of antiviral siRNA/miRNA pathways and inhibitory factors.

Endogenous miRNAs are transcribed from the genome as pri-miRNAs and are processed into pre-miRNAs by the Drosha/DGCR8 complex. Pre-miRNAs are exported to the cytoplasm and are processed into miRNA duplexes by Dicer and its partner protein complex. Viral RNAs are capable of being processed by the same ribonucleases; however, some VSRs or ISGs are reported to inhibit the processing of small RNAs in mammalian cells. Mature miRNAs or siRNAs silence both endogenous and exogenous RNAs loaded on AGO proteins in collaboration with various RNA silencing regulators for the antiviral functions. Endogenous nucleic acids and proteins: blue; viral nucleic acids and proteins: red. AGO, argonaute; dsRNA, double-stranded RNA; ISG, IFN-stimulated gene; miRNA, microRNA; pre-miRNA, precursor miRNA; pri-miRNA, primary transcript; siRNA, small interfering RNA; VSR, viral suppressor of RNAi.

https://doi.org/10.1371/journal.ppat.1010091.g001

Two types of mechanisms, those enabled by viral suppressors of RNAi (VSRs) and IFN signaling, reportedly limit the effectiveness of viral siRNAs in mammalian immunity. Several viral genes are reported as VSRs. The deletion or mutation of VSRs such as B2 protein of NoV [28] or nonstructural protein 1 (NS1) of influenza A virus (IAV) [29] leads to the production of virus-derived siRNAs (vsiRNAs) in human or mouse somatic cells, whereas infection by the intact viruses does not (Table 2) [2846]. Several proteins, such as virion protein 35 (VP35) of Ebola or Marburg virus, can prevent vsiRNA accumulation in cells infected by viruses lacking their native VSR, e.g., IAV lacking NS1 [31]. Furthermore, the deletion of these VSRs can repress viral replication, and the repression is recovered by Dicer or AGO2 deficiency. In addition, inhibitory peptides VSRs can induce the production of vsiRNAs [47], suggesting that the VSRs increase viral replication by inhibiting vsiRNA processing [41]. Human Dicer, however, also regulates the maturation of endogenous miRNAs, so Dicer deficiency may be assumed to affect posttranscriptional gene regulation by endogenous miRNAs as well, while AGO2 deficiency has little effect on the overall miRNA population. In other words, even in settings in which human Dicer has an antiviral effect, it is difficult to interpret whether this antiviral effect is derived from viral RNA cleavage by vsiRNAs or the regulation of antiviral gene expression by endogenous miRNAs. VSRs have been shown to act at various steps in antiviral RNAi pathways, including sequestration of dsRNA and inhibition of RNA silencing factors by RNA/protein or protein/protein interaction [30,45,46].

Antagonism between IFN and RNAi pathways may also limit the effectiveness of the latter. Deletion of either MAVS (also as known as IPS-1), an essential adaptor protein in IFN signaling triggered by RIG-I or MDA5, or IFNAR1, an IFN receptor on the cell surface, enables dsRNA-induced RNAi in MEF [48]. dsRNA-induced RNAi uncovered in MAVS-depleted MEF is again lost upon AGO2 depletion, indicating that IFN signaling masks AGO2-dependent, dsRNA-induced RNAi in mammalian somatic cells [48]. Some IFN-stimulated genes (ISGs) encoded in mammalian genomes might function as endogenous RNAi suppressors, as IFN treatment represses small RNA processing by Dicer in noninfected cells [48,49]. At least one ISG, LGP2, has been shown to repress Dicer activity [4951]. IFN stimulates hundreds of ISGs, and additional ISGs may also be involved in the suppression of RNAi. While IFN-elicited effects may block vsiRNA production in some settings, antagonism by IFN may not be the only explanation for restricted antiviral RNAi activity in mammals. For example, deletion of RIG-I or MDA5 abrogated IFN production but did not uncover vsiRNA production during HeLa cell infection by positive-sense RNA viruses Sindbis virus (SINV), yellow fever virus 17D vaccine strain (YFV17D), and coxsackievirus B3 (CBV3) [52], arguing that the suppressive activity of ISGs cannot be evoked as the reason vsiRNA are not produced under those conditions. Notably, infection by these viruses did not limit the ability of lentivirus-expressed shRNAs to decrease luciferase expression, arguing that these viruses do not encode a VSR effective under these conditions.

Several observations may harmonize the apparently discordant observations in this field. Whereas in general differentiated cells produce IFN upon viral infection or poly(I:C) treatment, some pluripotent or multipotent stem cells do not [53] and show attenuated response to exogenous IFN [54]. Pluripotent stem cells are able to process viral dsRNA into vsiRNA during EMCV or NoV infection [55], and the vsiRNA production is inhibited by B2 protein of NoV [56]. Mouse embryonic stem cells (mESCs) have an attenuated IFN response but show dsRNA-induced RNAi, whereas this activity is not detected after differentiation [48]. Mouse neural progenitor cells infected with Zika virus (ZIKV) or SINV showed production of vsiRNAs [57,58]. The recent discovery of alternative Dicer isoforms with antiviral activity in mouse and human stem cells extends this concept [59]. Notably, some ISGs are constitutively expressed in pluripotent or multipotent human stem cells, although these show different expression patterns in different tissue’s stem cells [60]. It is possible that some stem cells have antiviral RNAi activity, but others do not. In addition, when interpreting the inhibitory effect of IFN or ISGs for antiviral RNAi, it is important to consider whether global translational repression has been induced by protein kinase R (PKR) under the conditions used. PKR is activated by viral dsRNA and induces global translational repression [61,62], and activation of PKR can occur upstream and potentially independently of IFN signaling via RLRs [63]. It is reported that PKR is constitutively activated in Dicer-deficient mESCs and IFN production is elevated in Dicer-deficient mESCs compared to WT mESCs [59,64,65], suggesting that differential viral replication in Dicer-deficient mESCs results from the combination of viral RNA elimination by antiviral RNAi and PKR-induced translational repression of viral proteins used in viral replication. In the setting of PKR activation, IFN production may not necessarily correspond to high protein expression of ISGs. Intriguingly, several VSRs have also been reported to act as repressors of IFN production and repressors of PKR activation by interfering with its dsRNA-binding activity [29,66]. In summary, accumulation of dsRNA has various consequences that limit cellular permissiveness to virus replication. Interactions between dsRNA-responsive pathways preclude simple statements of the physiological relevance of mammalian vsiRNAs, especially those that would characterize vsiRNAs as standing apart from simultaneously induced pathways long recognized as antiviral, such as IFN.

Posttranscriptional gene regulation by miRNA-directed RNA silencing

miRNAs direct posttranscriptional gene regulation and, in so doing, often regulate antiviral responses in a manner consistent with this being among their evolved functions (Fig 1). miRNAs are approximately 22-nt endogenous noncoding RNAs [67]. In contrast to the roughly equivalent number of protein-coding genes [68], the human and mouse genomes encode 1,917 and 1,234 miRNA precursors (pre-miRNAs), respectively, which are processed into 2,656 or 1,978 mature miRNAs [69]. miRNAs exhibit expression patterns specific to each tissue [70], suggesting that miRNAs are involved in the complex gene regulatory networks characteristic of each tissue. miRNAs are transcribed from the genome as primary transcripts (pri-miRNAs) and are processed into pre-miRNAs by a Drosha/DGCR8 complex named microprocessor [71]. Pre-miRNAs are exported by Exportin-5/Ran-GTP to the cytoplasm and are processed into miRNA duplexes by Dicer [7274]. The miRNA biogenesis machinery is sometimes directly involved in antiviral defense; Dicer regulates both vsiRNA and miRNA production in virus-infected cells, and Drosha is reportedly able to cleave pri-miRNA-like structures in viral RNAs [75,76]. miRNAs do not induce RNA cleavage even if incorporated into AGO2 but instead selectively regulate RNAs by destabilizing them or repressing their translation. miRNAs recognize target RNAs by complementary base pairing at the position 2 to 7 or 2 to 8 from the 5′ end, called the seed region [77]. As the seed region is only 6 or 7 nt long, a single miRNA may repress hundreds of genes simultaneously [78].

Some miRNAs target endogenous mRNAs in a manner that promotes antiviral responses, while the others target viral RNAs directly (Table 3) [7987]. As examples of the former, miR-221/-222 increases after macrophage stimulation by tumor necrosis factor-α (TNF-α) and inhibits human immunodeficiency virus-1 (HIV-1) entry by down-regulating CD4 mRNA [79], and miR-3614, expressed in macrophages, increases after DENV infection and represses infection by targeting ADAR1 mRNA [80]. As an example of the latter, miR-323 increases after IAV infection and represses infection by targeting viral PB1 RNA [85].

thumbnail
Table 3. Examples of antiviral immunity directed by endogenous miRNAs.

https://doi.org/10.1371/journal.ppat.1010091.t003

As for siRNA, mutual regulation with the IFN system has also been reported for miRNA. IFN stimulation leads to distinct regulation of miRNA pools, with some increased and some decreased [84,87]. For example, IFN-induced miR-1/-30/-128/-196/-296/-351/-431/-448 display antiviral activity against hepatitis C virus (HCV), whereas miR-122, which is essential for HCV replication, is reduced by IFN [86,87]. miR-378 and -30e are repressed by IFN leading to activation of natural killer (NK) cells, enhancing their cytotoxicity [84]. In addition to changing abundance, epi-transcriptional modification of miRNAs can influence their antiviral activities. For example, human cytomegalovirus (HCMV) infection induces the expression of ADAR1 p110, which edits an adenosine in the seed of miR-376a to inosine [88]. This modifies the miRNA’s targeting and influences the susceptibility of infected cells to elimination by NK cells.

Both global regulation of miRNAs and regulation of specific populations of miRNAs may enhance virus elimination or link the innate and adaptive immune systems. Global decrease of miRNAs induces proinflammatory cytokine production [89]. Specific decrease in the group of miRNAs bound by TRBP up-regulates apoptosis regulatory genes and results in increased apoptosis of virus-infected cells [83]. For example, miR-106b targets several caspase mRNAs, including both initiator and executioner caspases, and its decrease causes relative up-regulation of these transcripts and enhances apoptosis of virus-infected cells [83]. miRNAs can also be released from infected cells and transported in extracellular vesicles (EVs), suggesting the possibility that miRNAs function like cytokines to transmit signals and modify gene expression of other cells in both a paracrine and autocrine manner [90]. Indeed, miRNAs have been shown to function as paracrine agonists by activating TLRs to promote IFN secretion [91,92], indicating that miRNA dynamics influence both intracellular and intercellular networks relevant to antiviral states. Like cytokine levels or antibody titers, tracking EV-packaged miRNAs has been proposed as a biomarker for prediction of antiviral efficacy [93]; for example, serum levels of miR-122 in patients infected with HCV reflects the success of IFN/ribavirin therapy [94]. Lastly, some viruses encode miRNAs; these are understood to counteract antiviral immunity and promote virus replication and are outside our scope but have been reviewed elsewhere [95].

CRISPR-like adaptive immunity directed by piRNAs

piRNAs are small RNAs that have been experimentally demonstrated to interact with Piwi proteins. piRNAs are often 24 to 31 nt, and most are transcribed from regions known as piRNA clusters. The biogenesis, nucleotide composition, and modification pattern of many piRNAs is distinct from other small RNAs (Fig 2). piRNAs can be generated in at least 2 different ways: phased pathways, in which piRNAs are sequentially produced from the same RNA strand, and ping-pong amplification, in which a pair of piRNAs are produced from dsRNA formed by the hybridization of a piRNA with its target [96]. Mature piRNAs form piRNA-induced silencing complexes (piRISCs) with Piwi proteins, and these complexes can induce heterochromatin formation or directly cleave their target RNA. As piRISC targets are often transcripts of transposable elements (TEs) and defects in the piRNA system often lead to gametogenesis failure, the canonical function of piRNAs is considered to be maintenance of the genome integrity of germ cells. So far, bona fide mammalian piRNAs have been found in the gonads, although Piwi proteins and/or small RNAs sharing features of piRNAs have been associated with functions in some somatic tissues, such as regulation of innate immunity in the lungs [97].

thumbnail
Fig 2. Overview of antiviral piRNA pathway and antiviral tRFs.

piRNAs with antiviral potential can be generated via several mechanisms: primary processing of piRNA cluster transcripts that contain an EVE, direct non-piRNA guided cleavage of viral RNA via a Piwi protein (vpiRNA), EVE-derived piRNA- or vpiRNA-guided cleavage of viral RNAs via a ping-pong amplification loop, or PIWI interactions with virus-homologous noncanonical piRNAs such as tRFs. EVE, endogenous viral element; piRNA, PIWI-interacting RNA; tRF, tRNA fragment; vpiRNA, virus-specific PIWI-interacting RNA.

https://doi.org/10.1371/journal.ppat.1010091.g002

The evolutionary origins and replication strategies of some viruses and TEs overlap; viruses and TEs can be considered together as mobile genetic elements, and recent reports in nonmammalian organisms suggest that both can be repressed by the piRNA system [98,99]. Sources for antiviral piRNAs include direct processing of exogenous viral RNAs [100], RNA fragments (e.g., tRNA; see below), or transcripts from endogenous viral elements (EVEs), including those within piRNA clusters. About 9% of a human’s genome is accounted for by EVEs, which are virus-derived sequences having undergone horizontal gene transfer (HGT) into the germline of our ancestors. EVEs, such as endogenous retroviruses (ERVs), may give rise to antiviral piRNAs. Koala retrovirus-A (KoRV-A) is found as both an infectious virus and as ERV provirus copies in some Koala germline genomes. Similarly, ecotropic murine leukemia viruses (MuLVs) recently entered some laboratory mouse germlines as ERVs and, in some cases, still encode particles capable of infecting mouse cells. Sense-stranded 24 to 31 nt testes-expressed small RNAs are derived from unspliced KoRV-A transcripts and may suppress this virus in the gonad, and sense-biased piRNAs made from ecotropic MuLV are similarly detected in the testes of mice whose genomes contain such proviruses [18]. Not all EVEs are derived from retroviruses; endogenous bornavirus-like nucleoprotein elements (EBLNs) are derived from an ancient relative of bornaviruses, which are negative-strand RNA viruses [101]. EBLNs entered rodent and primate lineages independently, yet EBLN-derived piRNAs are detected in gonads of both mouse and marmoset. This is remarkable given both the limited number of EBLNs and the small fraction of the genome that encodes piRNAs and likely indicates that EBLN integrations into piRNA clusters have been selectively retained [17]. Production of antiviral piRNAs from EVEs is reminiscent of an adaptive immune strategy in prokaryotes, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system [102]. In both cases, sequences from “non-self” mobile elements are incorporated into the genome by HGT to function in immune memory [103].

In many somatic or cancer cells, epigenetic manipulations that result in ERV expression also induce IFN signaling in an RLR-dependent manner [104,105]. In piRNA-deficient germ cells, ERV and other TE up-regulation is associated with meiotic arrest and apoptosis [106], though the precise signals triggering these defects remain unclear. Mouse germ cells express TLR3 and MDA5 and produce IFN and up-regulate ISGs upon poly(I:C) stimulation [107,108]. IFN overproduction in transgenic mice leads to male-specific sterility and increased apoptosis of spermatocytes [109]. An intact piRNA pathway may be necessary to prevent ERVs, inevitably expressed during the epigenetic erasures inherent to germ cell development, from triggering innate immune pathways incompatible with germ cell survival. Notably, in neural progenitor cells, TE expression upon DNA demethylation is met with a dramatic up-regulation of piRNA biogenesis genes [110], suggesting that some somatic cells may remain poised to generate piRNAs upon perturbation.

tRNAs and tRNA fragments (tRFs) as antiviral molecules

tRNAs regulate virus infections and TEs in addition to, or connected with, their canonical role in protein synthesis. Viruses generally do not encode tRNAs, while in humans, tRNAs are among the most abundant RNAs [111]. There are 10 times as many tRNA genes as codon–anticodon pairs, and considerable diversity exists in tRNA sequences besides the anticodon loop. Most tRNA genes cluster in discrete loci, the largest of which is adjacent to the major histocompatibility complex class I (MHC-I) coding region on chromosome 6 and encodes nearly all major tRNAs [112]. tRNAs are 70 to 90 nt in length and can be processed into tRFs (usually ranging from 14 to 32 nt) by ribonucleases including angiogenin and Dicer in a stimulus-dependent manner [111,113115]. tRFs derive from various points along the tRNA cloverleaf structure; 5′ and 3′ end-derived tRF halves (tRF-5 and tRF-3, respectively) have most often been reported to regulate viral infection.

Endogenous and exogenous retroviruses, as well as the TEs derived from them, use tRNAs as primers to initiate reverse transcription. IAP is an active mouse TE in the family of lysine tRNA-primed ERVs, and at least one IAP locus encodes functional membrane fusion machinery and can produce infectious particles [116]. tRFs were shown to inhibit IAP, as well as other TEs dependent on reverse transcription (MusD/ETn), by two mechanisms: posttranscriptional silencing and blocking interaction with tRNAs necessary for priming reverse transcription [117]. tRFs may indirectly influence replication of non-retroviruses; respiratory syncytial virus (RSV) infection increases tRF production in infected cells, and while one of the resulting tRFs can increase virus replication, the net effect of these changes on RSV infection outcome in cells remains unknown [118]. A tRF derived from the pre-tRNA-Ser 3′ trailer sequesters host RNA chaperone La/SSB and represses La/SSB-dependent viral gene expression during HCV infection [119]. While these reports suggest specific tRFs’ involvement in virus infection, global changes to tRNAs and tRFs are also linked to antiviral immunity; protecting tRNAs from stress-induced cleavage by angiogenin in T cells was recently shown to be an activity of schlafen 2, deletion of which results in greater susceptibility to viral infection [120]. This activity is reminiscent of the anti-HIV activity of schlafen 11, which is also dependent on regulation of the tRNA pool [121].

Some tRFs are reported to be loaded into Ago or Piwi proteins. tRF-5s and tRF-3s associate with all Ago paralogues in humans as tRNA-derived miRNAs (td-miRNA) [122]. Several have additionally been shown to guide sequence-specific silencing of cellular [123] and viral [124] transcripts. tRF halves serve as a major source of tRNA-derived piRNA (td-piRNA) in silkworm [125], but the biogenesis of td-piRNAs in mammals remains to be fully clarified. Gtsf1, the murine homologue of drosophila Asterix, is a piRNA biogenesis factor that preferentially binds tRNA over piRNA and may suppress tRNA-primed retroelements [126], although the mechanism remains incompletely defined. Several tRFs are reported to bind Piwil4 (Hiwi2) in both normal and transformed human cells [127]. PIWIL2 (HILI) has been reported to interact with intact tRNAs and induce translational repression of HIV-1 via this activity [128], though HILI may also interact with tRFs that were undetected using the methods applied [129].

Current and future applications of small RNAs as nucleic acid medicine

Small RNAs have favorable characteristics as antiviral therapeutics. Various types of 18 to 30 nt length RNAs have been approved by the FDA as nucleic acid medicines as of April 2021: 9 antisense oligonucleotides (ASOs), 1 aptamer, 4 siRNAs, and 1 CpG oligonucleotide. The specific sequences and modifications of these nucleic acid medicines are not present in nature, but each harnesses an endogenous system directed by small RNAs. As powerfully demonstrated in development of mRNA vaccines against COVID-19, which took only 45 days from the publication of the SARS-CoV-2 draft genome sequence to shipment of a product for clinical trials, nucleic acid medicines can be chemically synthesized in large amounts quickly. Target and off-target recognition of small RNAs can be rationally designed and screened in silico against databases of nucleic acids present in uninfected human cells. This stands as an advantage over existing platforms for designing protein-based therapeutics, notably monoclonal antibodies, despite advances in structure prediction and molecular dynamic simulations. Among the approved small RNA medicines, ASO and siRNA were rationally designed based on predicted complementarity to the target RNA, whereas the sequence of the approved aptamer was experimentally screened using SELEX [130]. Among the small RNAs reviewed in previous sections, only siRNAs have been approved as medicines; miRNA, piRNA, and tRNA-based medicines remain a therapeutic frontier. Delivery of nucleic acid medicines to their target tissues was a major early hurdle; small RNAs are quickly degraded in the serum by RNases or subject to glomerular filtration. However, recent advances in drug delivery systems such as lipid nanoparticles or nucleic acid glycosylation have addressed these challenges, as mRNA vaccination has again demonstrated. Understanding how small RNAs, such as EV-packaged miRNAs, act as paracrine regulators in physiological mammalian immunity holds promise for additional improvements. The personalizability of nucleic acids is another promising feature for individualized medicine. For example, a personalized ASO, Milasen, was designed and used for a patient with Batten disease. This required less than a year from sequencing the patient’s genome, which enabled rational design, to symptom improvement [131]. Similar to human genome sequencing, which is transitioning to routine clinical use, virus sequencing infrastructure will substantially increase as a consequence of the current pandemic, so designing small RNAs based on the viral genotypes circulating in an individual or population is becoming more realistic. Similar to those in development for cancer, small RNAs targeting known pathogenic mutations might be used to anticipate and limit viral evolution [132]. Harnessing the piRNA pathway for antiviral therapy would be complicated by the limited expression of Piwi proteins in somatic cells, yet the possibility of “looped” production between the delivered nucleic acid medicine and a target viral quasispecies is also notable with respect to evolution of resistance. Compared to delivery of Cas9 ribonucleoproteins, which is now clinically applicable [133], delivery of human Piwi ribonucleoproteins would carry the theoretical advantage of immune tolerance to the endonuclease component and could potentially be coupled to “endogenous-inspired” delivery systems [134].

Conclusions

Uncovering the mechanisms that enable human immunity to viruses has led to many success stories in development of antiviral therapies, from plant-derived compounds to IFN therapy to CCR5 antagonists to monoclonal antibodies. Clarifying the role of small RNA–directed antiviral immune systems in mammals will likely provide additional successes; the recently described antiviral actions of miRNAs, piRNAs, and tRNAs are especially promising. In parallel to advances in the basic science, mRNA-based therapies and prophylactics have seen accelerated development since the SARS-CoV-2 pandemic, seemingly overcoming previous barriers to nucleic acid medicines. Small RNAs have big potential for the future therapeutic applications.

References

  1. 1. Suttle CA. Viruses in the sea. Nature. 2005;437(7057):356–61. pmid:16163346
  2. 2. Watanabe T, Suzuki N, Tomonaga K, Sawa H, Matsuura Y, Kawaguchi Y, et al. Neo-virology: The raison d’etre of viruses. Virus Res. 2019;274:197751. pmid:31520652
  3. 3. Westra ER, Levin BR. It is unclear how important CRISPR-Cas systems are for protecting natural populations of bacteria against infections by mobile genetic elements. Proc Natl Acad Sci U S A. 2020;117(45):27777–85. pmid:33122438
  4. 4. Buchmann K. Evolution of Innate Immunity: Clues from Invertebrates via Fish to Mammals. Front Immunol. 2014;5:459. pmid:25295041
  5. 5. Hirano M, Das S, Guo P, Cooper MD. The evolution of adaptive immunity in vertebrates. Adv Immunol. 2011;109:125–57. pmid:21569914
  6. 6. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 2001;413(6857):732–8. pmid:11607032
  7. 7. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol. 2004;5(7):730–7. pmid:15208624
  8. 8. Fitzgerald KA, Kagan JC. Toll-like Receptors and the Control of Immunity. Cell. 2020;180(6):1044–66. pmid:32164908
  9. 9. Rehwinkel J, Gack MU. RIG-I-like receptors: their regulation and roles in RNA sensing. Nat Rev Immunol. 2020;20(9):537–51. pmid:32203325
  10. 10. Tassetto M, Kunitomi M, Andino R. Circulating Immune Cells Mediate a Systemic RNAi-Based Adaptive Antiviral Response in Drosophila. Cell. 2017;169(2):314–25.e13. pmid:28388413
  11. 11. Hambleton S, Goodbourn S, Young DF, Dickinson P, Mohamad SM, Valappil M, et al. STAT2 deficiency and susceptibility to viral illness in humans. Proc Natl Acad Sci U S A. 2013;110(8):3053–8. pmid:23391734
  12. 12. Keeling DM, Garza P, Nartey CM, Carvunis AR. The meanings of ’function’ in biology and the problematic case of de novo gene emergence. Elife. 2019;8. pmid:31674305
  13. 13. Maillard PV, van der Veen AG, Poirier EZ, Reis e Sousa C. Slicing and dicing viruses: antiviral RNA interference in mammals. EMBO J. 2019;38(8). pmid:30872283
  14. 14. Girardi E, López P, Pfeffer S. On the Importance of Host MicroRNAs During Viral Infection. Front Genet. 2018;9:439. pmid:30333857
  15. 15. Takahashi T, Ui-Tei K. Mutual Regulation of RNA Silencing and the IFN Response as an Antiviral Defense System in Mammalian Cells. Int J Mol Sci. 2020;21(4). pmid:32079277
  16. 16. Yu X, Xie Y, Zhang S, Song X, Xiao B, Yan Z. tRNA-derived fragments: Mechanisms underlying their regulation of gene expression and potential applications as therapeutic targets in cancers and virus infections. Theranostics. 2021;11(1):461–9. pmid:33391486
  17. 17. Parrish NF, Fujino K, Shiromoto Y, Iwasaki YW, Ha H, Xing J, et al. piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals. RNA. 2015;21(10):1691–703. pmid:26283688
  18. 18. Yu T, Koppetsch BS, Pagliarani S, Johnston S, Silverstein NJ, Luban J, et al. The piRNA Response to Retroviral Invasion of the Koala Genome. Cell. 2019;179(3):632–43.e12. pmid:31607510
  19. 19. Eigen M, Lindemann BF, Tietze M, Winkler-Oswatitsch R, Dress A, von Haeseler A. How old is the genetic code? Statistical geometry of tRNA provides an answer. Science. 1989;244(4905):673–9. pmid:2497522
  20. 20. Özata DM, Yu T, Mou H, Gainetdinov I, Colpan C, Cecchini K, et al. Evolutionarily conserved pachytene piRNA loci are highly divergent among modern humans. Nat Ecol Evol. 2020;4(1):156–68. pmid:31900453
  21. 21. Kaufmann SHE. Immunology’s Coming of Age. Front Immunol. 2019;10:684. pmid:31001278
  22. 22. Zhang R, Jing Y, Zhang H, Niu Y, Liu C, Wang J, et al. Comprehensive Evolutionary Analysis of the Major RNA-Induced Silencing Complex Members. Sci Rep. 2018;8(1):14189. pmid:30242207
  23. 23. Wang MB, Metzlaff M. RNA silencing and antiviral defense in plants. Curr Opin Plant Biol. 2005;8(2):216–22. pmid:15753004
  24. 24. Leggewie M, Schnettler E. RNAi-mediated antiviral immunity in insects and their possible application. Curr Opin Virol. 2018;32:108–14. pmid:30428425
  25. 25. Guo Z, Li Y, Ding SW. Small RNA-based antimicrobial immunity. Nat Rev Immunol. 2019;19(1):31–44. pmid:30301972
  26. 26. Adiliaghdam F, Basavappa M, Saunders TL, Harjanto D, Prior JT, Cronkite DA, et al. A Requirement for Argonaute 4 in Mammalian Antiviral Defense. Cell Rep. 2020;30(6):1690–701.e4. pmid:32049003
  27. 27. Han Q, Chen G, Wang J, Jee D, Li WX, Lai EC, et al. Mechanism and Function of Antiviral RNA Interference in Mice. mBio. 2020;11(4). pmid:32753500
  28. 28. Sullivan CS, Ganem D. A virus-encoded inhibitor that blocks RNA interference in mammalian cells. J Virol. 2005;79(12):7371–9. pmid:15919892
  29. 29. Li WX, Li H, Lu R, Li F, Dus M, Atkinson P, et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc Natl Acad Sci U S A. 2004;101(5):1350–5. pmid:14745017
  30. 30. Haasnoot J, de Vries W, Geutjes EJ, Prins M, de Haan P, Berkhout B. The Ebola virus VP35 protein is a suppressor of RNA silencing. PLoS Pathog. 2007;3(6):e86. pmid:17590081
  31. 31. Li Y, Basavappa M, Lu J, Dong S, Cronkite DA, Prior JT, et al. Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells. Nat Microbiol. 2016;2:16250. pmid:27918527
  32. 32. Bennasser Y, Le SY, Benkirane M, Jeang KT. Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity. 2005;22(5):607–19. pmid:15894278
  33. 33. Chinnappan M, Singh AK, Kakumani PK, Kumar G, Rooge SB, Kumari A, et al. Key elements of the RNAi pathway are regulated by hepatitis B virus replication and HBx acts as a viral suppressor of RNA silencing. Biochem J. 2014;462(2):347–58. pmid:24902849
  34. 34. Wang Y, Kato N, Jazag A, Dharel N, Otsuka M, Taniguchi H, et al. Hepatitis C virus core protein is a potent inhibitor of RNA silencing-based antiviral response. Gastroenterology. 2006;130(3):883–92. pmid:16530526
  35. 35. Zhou H, Qian Q, Shu T, Xu J, Kong J, Mu J, et al. Hepatitis C Virus NS2 Protein Suppresses RNA Interference in Cells. Virol Sin. 2020;35(4):436–44. pmid:31777009
  36. 36. Qian Q, Zhou H, Shu T, Mu J, Fang Y, Xu J, et al. The Capsid Protein of Semliki Forest Virus Antagonizes RNA Interference in Mammalian Cells. J Virol. 2020;94(3). pmid:31694940
  37. 37. Fabozzi G, Nabel CS, Dolan MA, Sullivan NJ. Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway. J Virol. 2011;85(6):2512–23. pmid:21228243
  38. 38. Kakumani PK, Ponia SS, et al. Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor. J Virol. 2013;87(16):8870–83. pmid:23741001
  39. 39. Kakumani PK, Rajgokul KS, Ponia SS, Kaur I, Mahanty S, Medigeshi GR, et al. Dengue NS3, an RNAi suppressor, modulates the human miRNA pathways through its interacting partner. Biochem J. 2015;471(1):89–99. pmid:26221025
  40. 40. Qiu Y, Xu YP, Wang M, Miao M, Zhou H, Xu J, et al. Flavivirus induces and antagonizes antiviral RNA interference in both mammals and mosquitoes. Sci Adv. 2020;6(6):eaax7989. pmid:32076641
  41. 41. Qiu Y, Xu Y, Zhang Y, Zhou H, Deng YQ, Li XF, et al. Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals. Immunity. 2017;46(6):992–1004.e5. pmid:28636969
  42. 42. Cui L, Wang H, Ji Y, Yang J, Xu S, Huang X, et al. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells. J Virol. 2015;89(17):9029–43. pmid:26085159
  43. 43. Karjee S, Minhas A, Sood V, Ponia SS, Banerjea AC, Chow VT, et al. The 7a accessory protein of severe acute respiratory syndrome coronavirus acts as an RNA silencing suppressor. J Virol. 2010;84(19):10395–401. pmid:20631126
  44. 44. Mu J, Xu J, Zhang L, Shu T, Wu D, Huang M, et al. SARS-CoV-2-encoded nucleocapsid protein acts as a viral suppressor of RNA interference in cells. Sci China Life Sci. 2020;63(9):1413–6. pmid:32291557
  45. 45. Lu S, Cullen BR. Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and MicroRNA biogenesis. J Virol. 2004;78(23):12868–76. pmid:15542639
  46. 46. Andersson MG, Haasnoot PC, Xu N, Berenjian S, Berkhout B, Akusjärvi G. Suppression of RNA interference by adenovirus virus-associated RNA. J Virol. 2005;79(15):9556–65. pmid:16014917
  47. 47. Fang Y, Liu Z, Qiu Y, Kong J, Fu Y, Liu Y, et al. Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviral infection in vivo. Immunity. 2021;54(10):2231–44.e6. pmid:34555337
  48. 48. Maillard PV, Van der Veen AG, Deddouche-Grass S, Rogers NC, Merits A, Reis e Sousa C. Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells. EMBO J. 2016;35(23):2505–18. pmid:27815315
  49. 49. Takahashi T, Nakano Y, Onomoto K, Murakami F, Komori C, Suzuki Y, et al. LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs. Nucleic Acids Res. 2018;46(17):9134–47. pmid:29939295
  50. 50. van der Veen AG, Maillard PV, Schmidt JM, Lee SA, Deddouche-Grass S, Borg A, et al. The RIG-I-like receptor LGP2 inhibits Dicer-dependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells. EMBO J. 2018;37(4). pmid:29351913
  51. 51. Zhang Y, Xu Y, Dai Y, Li Z, Wang J, Ye Z, et al. Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2. PLoS Pathog. 2021;17(8):e1009790. pmid:34343211
  52. 52. Schuster S, Tholen LE, Overheul GJ, van Kuppeveld FJM, van Rij RP. Deletion of Cytoplasmic Double-Stranded RNA Sensors Does Not Uncover Viral Small Interfering RNA Production in Human Cells. mSphere. 2017;2(4).
  53. 53. Burke DC, Graham CF, Lehman JM. Appearance of interferon inducibility and sensitivity during differentiation of murine teratocarcinoma cells in vitro. Cell. 1978;13(2):243–8. pmid:627035
  54. 54. Hong XX, Carmichael GG. Innate immunity in pluripotent human cells: attenuated response to interferon-β. J Biol Chem. 2013;288(22):16196–205. pmid:23599426
  55. 55. Maillard PV, Ciaudo C, Marchais A, Li Y, Jay F, Ding SW, et al. Antiviral RNA interference in mammalian cells. Science. 2013;342(6155):235–8. pmid:24115438
  56. 56. Li Y, Lu J, Han Y, Fan X, Ding SW. RNA interference functions as an antiviral immunity mechanism in mammals. Science. 2013;342(6155):231–4. pmid:24115437
  57. 57. Xu YP, Qiu Y, Zhang B, Chen G, Chen Q, Wang M, et al. Zika virus infection induces RNAi-mediated antiviral immunity in human neural progenitors and brain organoids. Cell Res. 2019;29(4):265–73. pmid:30814679
  58. 58. Zhang Y, Li Z, Ye Z, Xu Y, Wang B, Wang C, et al. The activation of antiviral RNA interference not only exists in neural progenitor cells but also in somatic cells in mammals. Emerg Microbes Infect. 2020;9(1):1580–9. pmid:32576094
  59. 59. Poirier EZ, Buck MD, Chakravarty P, Carvalho J, Frederico B, Cardoso A, et al. An isoform of Dicer protects mammalian stem cells against multiple RNA viruses. Science. 2021;373(6551):231–6. pmid:34244417
  60. 60. Wu X, Dao Thi VL, Huang Y, Billerbeck E, Saha D, Hoffmann HH, et al. Intrinsic Immunity Shapes Viral Resistance of Stem Cells. Cell. 2018;172(3):423–38.e25. pmid:29249360
  61. 61. Farrell PJ, Sen GC, Dubois MF, Ratner L, Slattery E, Lengyel P. Interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded RNA. Proc Natl Acad Sci U S A. 1978;75(12):5893–7. pmid:282611
  62. 62. Munir M, Berg M. The multiple faces of protein kinase R in antiviral defense. Virulence. 2013;4(1):85–9. pmid:23314571
  63. 63. Onomoto K, Jogi M, Yoo JS, Narita R, Morimoto S, Takemura A, et al. Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity. PLoS ONE. 2012;7(8):e43031. pmid:22912779
  64. 64. Witteveldt J, Knol LI, Macias S. MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response. elife. 2019;8. pmid:31012846
  65. 65. Gurung C, Fendereski M, Sapkota K, Guo J, Huang F, Guo YL. Dicer represses the interferon response and the double-stranded RNA-activated protein kinase pathway in mouse embryonic stem cells. J Biol Chem. 2021;296:100264. pmid:33837743
  66. 66. Basler CF, Wang X, Mühlberger E, Volchkov V, Paragas J, Klenk HD, et al. The Ebola virus VP35 protein functions as a type I IFN antagonist. Proc Natl Acad Sci U S A. 2000;97(22):12289–94. pmid:11027311
  67. 67. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281–97. pmid:14744438
  68. 68. Howe KL, Achuthan P, Allen J, Alvarez-Jarreta J, Amode MR, Armean IM, et al. Ensembl 2021. Nucleic Acids Res. 2021;49(D1):D884–D91. pmid:33137190
  69. 69. Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 2014;42(Database issue):D68–73. pmid:24275495
  70. 70. Ludwig N, Leidinger P, Becker K, Backes C, Fehlmann T, Pallasch C, et al. Distribution of miRNA expression across human tissues. Nucleic Acids Res. 2016;44(8):3865–77. pmid:26921406
  71. 71. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425(6956):415–9. pmid:14508493
  72. 72. Lund E, Güttinger S, Calado A, Dahlberg JE, Kutay U. Nuclear export of microRNA precursors. Science. 2004;303(5654):95–8. pmid:14631048
  73. 73. Yi R, Qin Y, Macara IG, Cullen BR. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 2003;17(24):3011–6. pmid:14681208
  74. 74. Doi N, Zenno S, Ueda R, Ohki-Hamazaki H, Ui-Tei K, Saigo K. Short-interfering-RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors. Curr Biol 2003;13(1):41–6. pmid:12526743
  75. 75. Shapiro JS, Schmid S, Aguado LC, Sabin LR, Yasunaga A, Shim JV, et al. Drosha as an interferon-independent antiviral factor. Proc Natl Acad Sci U S A. 2014;111(19):7108–13. pmid:24778219
  76. 76. Aguado LC, Schmid S, May J, Sabin LR, Panis M, Blanco-Melo D, et al. RNase III nucleases from diverse kingdoms serve as antiviral effectors. Nature. 2017;547(7661):114–7. pmid:28658212
  77. 77. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell. 2003;115(7):787–98. pmid:14697198
  78. 78. Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature. 2005;433(7027):769–73. pmid:15685193
  79. 79. Lodge R, Ferreira Barbosa JA, Lombard-Vadnais F, Gilmore JC, Deshiere A, Gosselin A, et al. Host MicroRNAs-221 and -222 Inhibit HIV-1 Entry in Macrophages by Targeting the CD4 Viral Receptor. Cell Rep. 2017;21(1):141–53. pmid:28978468
  80. 80. Diosa-Toro M, Echavarría-Consuegra L, Flipse J, Fernández GJ, Kluiver J, van den Berg A, et al. MicroRNA profiling of human primary macrophages exposed to dengue virus identifies miRNA-3614-5p as antiviral and regulator of ADAR1 expression. PLoS Negl Trop Dis. 2017;11(10):e0005981. pmid:29045406
  81. 81. Singaravelu R, Ahmed N, Quan C, Srinivasan P, Ablenas CJ, Roy DG, et al. A conserved miRNA-183 cluster regulates the innate antiviral response. J Biol Chem. 2019;294(51):19785–94. pmid:31694919
  82. 82. Zhao L, Zhang X, Wu Z, Huang K, Sun X, Chen H, et al. The Downregulation of MicroRNA hsa-miR-340-5p in IAV-Infected A549 Cells Suppresses Viral Replication by Targeting RIG-I and OAS2. Mol Ther Nucleic Acids. 2019;14:509–19. pmid:30753994
  83. 83. Takahashi T, Nakano Y, Onomoto K, Yoneyama M, Ui-Tei K. LGP2 virus sensor enhances apoptosis by upregulating apoptosis regulatory genes through TRBP-bound miRNAs during viral infection. Nucleic Acids Res. 2020;48(3):1494–507. pmid:31799626
  84. 84. Wang P, Gu Y, Zhang Q, Han Y, Hou J, Lin L, et al. Identification of resting and type I IFN-activated human NK cell miRNomes reveals microRNA-378 and microRNA-30e as negative regulators of NK cell cytotoxicity. J Immunol. 2012;189(1):211–21. pmid:22649192
  85. 85. Song L, Liu H, Gao S, Jiang W, Huang W. Cellular microRNAs inhibit replication of the H1N1 influenza A virus in infected cells. J Virol. 2010;84(17):8849–60. pmid:20554777
  86. 86. Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science. 2005;309(5740):1577–81. pmid:16141076
  87. 87. Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature. 2007;449(7164):919–22. pmid:17943132
  88. 88. Nachmani D, Zimmermann A, Oiknine Djian E, Weisblum Y, Livneh Y, Khanh Le VT, et al. MicroRNA editing facilitates immune elimination of HCMV infected cells. PLoS Pathog. 2014;10(2):e1003963. pmid:24586166
  89. 89. Aguado LC, Schmid S, Sachs D, Shim JV, Lim JK, tenOever BR. microRNA Function Is Limited to Cytokine Control in the Acute Response to Virus Infection. Cell Host Microbe. 2015;18(6):714–22. pmid:26651947
  90. 90. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654–9. pmid:17486113
  91. 91. Fabbri M, Paone A, Calore F, Galli R, Gaudio E, Santhanam R, et al. MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response. Proc Natl Acad Sci U S A. 2012;109(31):E2110–6. pmid:22753494
  92. 92. Salvi V, Gianello V, Busatto S, Bergese P, Andreoli L, D’Oro U, et al. Exosome-delivered microRNAs promote IFN-α secretion by human plasmacytoid DCs via TLR7. JCI. Insight. 2018;3(10). pmid:29769437
  93. 93. Tribolet L, Kerr E, Cowled C, Bean AGD, Stewart CR, Dearnley M, et al. MicroRNA Biomarkers for Infectious Diseases: From Basic Research to Biosensing. Front Microbiol. 2020;11:1197. pmid:32582115
  94. 94. Köberle V, Waidmann O, Kronenberger B, Andrei A, Susser S, Füller C, et al. Serum microRNA-122 kinetics in patients with chronic hepatitis C virus infection during antiviral therapy. J Viral Hepat. 2013;20(8):530–5. pmid:23808991
  95. 95. Cullen BR. MicroRNAs as mediators of viral evasion of the immune system. Nat Immunol. 2013;14(3):205–10. pmid:23416678
  96. 96. Iwasaki YW, Siomi MC, Siomi H. PIWI-Interacting RNA: Its Biogenesis and Functions. Annu Rev Biochem. 2015;84:405–33. pmid:25747396
  97. 97. Wasserman GA, Szymaniak AD, Hinds AC, Yamamoto K, Kamata H, Smith NM, et al. Expression of Piwi protein MIWI2 defines a distinct population of multiciliated cells. J Clin Invest. 2017;127(10):3866–76. pmid:28920925
  98. 98. Sun YH, Xie LH, Zhuo X, Chen Q, Ghoneim D, Zhang B, et al. Domestic chickens activate a piRNA defense against avian leukosis virus. Elife. 2017;6. pmid:28384097
  99. 99. Suzuki Y, Baidaliuk A, Miesen P, Frangeul L, Crist AB, Merkling SH, et al. Non-retroviral Endogenous Viral Element Limits Cognate Virus Replication in Aedes aegypti Ovaries. Curr Biol. 2020;30(18):3495–506.e6. pmid:32679098
  100. 100. Miesen P, Joosten J, van Rij RP. PIWIs Go Viral: Arbovirus-Derived piRNAs in Vector Mosquitoes. PLoS Pathog. 2016;12(12):e1006017. pmid:28033427
  101. 101. Horie M, Honda T, Suzuki Y, Kobayashi Y, Daito T, Oshida T, et al. Endogenous non-retroviral RNA virus elements in mammalian genomes. Nature. 2010;463(7277):84–7. pmid:20054395
  102. 102. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315(5819):1709–12. pmid:17379808
  103. 103. Ophinni Y, Palatini U, Hayashi Y, Parrish NF. piRNA-Guided CRISPR-like Immunity in Eukaryotes. Trends Immunol. 2019;40(11):998–1010. pmid:31679813
  104. 104. Chiappinelli KB, Strissel PL, Desrichard A, Li H, Henke C, Akman B, et al. Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses. Cell. 2015;162(5):974–86. pmid:26317466
  105. 105. Kato M, Takemoto K, Shinkai Y. A somatic role for the histone methyltransferase Setdb1 in endogenous retrovirus silencing. Nat Commun. 2018;9(1):1683. pmid:29703894
  106. 106. Newkirk SJ, Lee S, Grandi FC, Gaysinskaya V, Rosser JM, Vanden Berg N, et al. Intact piRNA pathway prevents L1 mobilization in male meiosis. Proc Natl Acad Sci U S A. 2017;114(28):E5635–E44. pmid:28630288
  107. 107. Wang T, Zhang X, Chen Q, Deng T, Zhang Y, Li N, et al. Toll-like receptor 3-initiated antiviral responses in mouse male germ cells in vitro. Biol Reprod. 2012;86(4):106. pmid:22262694
  108. 108. Melaine N, Liénard MO, Guillaume E, Ruffault A, Dejucq-Rainsford N, Jégou B. Production of the antiviral proteins 2′5’oligoadenylate synthetase, PKR and Mx in interstitial cells and spermatogonia. J Reprod Immunol. 2003;59(1):53–60. pmid:12892903
  109. 109. Satie AP, Mazaud-Guittot S, Seif I, Mahé D, He Z, Jouve G, et al. Excess type I interferon signaling in the mouse seminiferous tubules leads to germ cell loss and sterility. J Biol Chem. 2011;286(26):23280–95. pmid:21515676
  110. 110. Jönsson ME, Ludvik Brattås P, Gustafsson C, Petri R, Yudovich D, Pircs K, et al. Activation of neuronal genes via LINE-1 elements upon global DNA demethylation in human neural progenitors. Nat Commun. 2019;10(1):3182. pmid:31320637
  111. 111. Cole C, Sobala A, Lu C, Thatcher SR, Bowman A, Brown JW, et al. Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. RNA. 2009;15(12):2147–60. pmid:19850906
  112. 112. Mungall AJ, Palmer SA, Sims SK, Edwards CA, Ashurst JL, Wilming L, et al. The DNA sequence and analysis of human chromosome 6. Nature. 2003;425(6960):805–11. pmid:14574404
  113. 113. Lee YS, Shibata Y, Malhotra A, Dutta A. A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev. 2009;23(22):2639–49. pmid:19933153
  114. 114. Su Z, Kuscu C, Malik A, Shibata E, Dutta A. Angiogenin generates specific stress-induced tRNA halves and is not involved in tRF-3-mediated gene silencing. J Biol Chem. 2019;294(45):16930–41. pmid:31582561
  115. 115. Kuscu C, Kumar P, Kiran M, Su Z, Malik A, Dutta A. tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner. RNA. 2018;24(8):1093–105. pmid:29844106
  116. 116. Ribet D, Harper F, Dupressoir A, Dewannieux M, Pierron G, Heidmann T. An infectious progenitor for the murine IAP retrotransposon: emergence of an intracellular genetic parasite from an ancient retrovirus. Genome Res. 2008;18(4):597–609. pmid:18256233
  117. 117. Schorn AJ, Gutbrod MJ, LeBlanc C, Martienssen R. LTR-Retrotransposon Control by tRNA-Derived Small RNAs. Cell. 2017;170(1):61–71.e11. pmid:28666125
  118. 118. Wang Q, Lee I, Ren J, Ajay SS, Lee YS, Bao X. Identification and functional characterization of tRNA-derived RNA fragments (tRFs) in respiratory syncytial virus infection. Mol Ther. 2013;21(2):368–79. pmid:23183536
  119. 119. Cho H, Lee W, Kim GW, Lee SH, Moon JS, Kim M, et al. Regulation of La/SSB-dependent viral gene expression by pre-tRNA 3′ trailer-derived tRNA fragments. Nucleic Acids Res. 2019;47(18):9888–901. pmid:31504775
  120. 120. Yue T, Zhan X, Zhang D, Jain R, Wang KW, Choi JH, et al. SLFN2 protection of tRNAs from stress-induced cleavage is essential for T cell-mediated immunity. Science. 2021;372(6543). pmid:33986151
  121. 121. Li M, Kao E, Gao X, Sandig H, Limmer K, Pavon-Eternod M, et al. Codon-usage-based inhibition of HIV protein synthesis by human schlafen 11. Nature. 2012;491(7422):125–8. pmid:23000900
  122. 122. Kumar P, Anaya J, Mudunuri SB, Dutta A. Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets. BMC Biol. 2014;12:78. pmid:25270025
  123. 123. Maute RL, Schneider C, Sumazin P, Holmes A, Califano A, Basso K, et al. tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma. Proc Natl Acad Sci U S A. 2013;110(4):1404–9. pmid:23297232
  124. 124. Yeung ML, Bennasser Y, Watashi K, Le SY, Houzet L, Jeang KT. Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid. Nucleic Acids Res. 2009;37(19):6575–86. pmid:19729508
  125. 125. Honda S, Kawamura T, Loher P, Morichika K, Rigoutsos I, Kirino Y. The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells. Nucleic Acids Res. 2017;45(15):9108–20. pmid:28645172
  126. 126. Ipsaro JJ, O’Brien PA, Bhattacharya S, Palmer AG, Joshua-Tor L. Asterix/Gtsf1 links tRNAs and piRNA silencing of retrotransposons. Cell Rep. 2021;34(13):108914. pmid:33789107
  127. 127. Keam SP, Young PE, McCorkindale AL, Dang TH, Clancy JL, Humphreys DT, et al. The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells. Nucleic Acids Res. 2014;42(14):8984–95. pmid:25038252
  128. 128. Peterlin BM, Liu P, Wang X, Cary D, Shao W, Leoz M, et al. Hili Inhibits HIV Replication in Activated T Cells. J Virol. 2017;91(11). pmid:28331090
  129. 129. Shigematsu M, Kawamura T, Morichika K, Izumi N, Kiuchi T, Honda S, et al. RNase κ promotes robust piRNA production by generating 2′,3′-cyclic phosphate-containing precursors. Nat Commun. 2021;12(1):4498. pmid:34301931
  130. 130. Ruckman J, Green LS, Beeson J, Waugh S, Gillette WL, Henninger DD, et al. 2’-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem. 1998;273(32):20556–67. pmid:9685413
  131. 131. Kim J, Hu C, Moufawad El Achkar C, Black LE, Douville J, Larson A, et al. Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease. N Engl J Med. 2019;381(17):1644–52. pmid:31597037
  132. 132. Acunzo M, Romano G, Nigita G, Veneziano D, Fattore L, Laganà A, et al. Selective targeting of point-mutated KRAS through artificial microRNAs. Proc Natl Acad Sci U S A. 2017;114(21):E4203–E12. pmid:28484014
  133. 133. Gillmore JD, Gane E, Taubel J, Kao J, Fontana M, Maitland ML, et al. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N Engl J Med. 2021;385(6):493–502. pmid:34215024
  134. 134. Segel M, Lash B, Song J, Ladha A, Liu CC, Jin X, et al. Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery. Science. 2021;373(6557):882–9. pmid:34413232