ABSTRACT
The question of whether any mammalian cells are able to mount an effective RNA interference-mediated antiviral innate immune response has remained highly controversial. In this Gem, I review recent data addressing this important issue and propose a testable hypothesis that can explain many of the apparently contradictory results published in this area of research.
INTRODUCTION
As originally defined by Fire et al. in 1998 (1), RNA interference (RNAi) is a cellular response whereby introduction of long, perfect double-stranded RNAs (dsRNAs) into cells results in the posttranscriptional inhibition of endogenous mRNAs that are perfectly complementary to the dsRNAs. We now know that the injected dsRNAs are processed by the cytoplasmic RNase III enzyme Dicer into ∼22-bp dsRNA duplexes, called small interfering RNAs (siRNAs) (2). One strand of the siRNA duplex is then essentially randomly loaded into the RNA-induced silencing complex RISC, where it serves as a guide RNA to direct RISC to complementary mRNA targets. Once bound, the Argonaut (Ago) component of RISC induces endonucleolytic cleavage of the mRNA, leading to mRNA degradation. A process comparable to RNAi had been previously identified in plants, where it was referred to as posttranscriptional gene silencing, and in some fungi, where it was termed quelling, so it seemed possible that the potential to mount an RNAi response might be a characteristic shared by all eukaryotic organisms, including mammals.
So why did RNAi evolve? RNAi could of course be used to regulate host cell gene expression, and in fact, it soon became clear that genomically encoded small RNAs very similar to siRNAs, called microRNAs (miRNAs), are present in the cells of all metazoan species. However, the more likely explanation for the existence of RNAi is that it initially evolved as an antiviral innate immune response, and some of the first siRNAs to be described were detected at high levels in plants infected with potato virus X (3). Importantly, all RNA viruses except retroviruses generate long, perfect dsRNA intermediates during replication of their RNA genome, and even dsDNA viruses generate high levels of perfect dsRNAs due to convergent transcription of both strands of their DNA genome. Such long, perfect dsRNAs, which are not normally found in uninfected cells, therefore function not only as potential substrates for siRNA biogenesis but also as an important pathogen-associated molecular pattern (PAMP) that alerts eukaryotic cells to the presence of an invading virus. Could RNAi represent a common innate antiviral defense in a wide range of multicellular eukaryotes?
As noted above, there is compelling evidence to support a key role for RNAi in antiviral immunity in plants, including the demonstration of high levels of siRNAs of viral origin in virus-infected cells, the greatly elevated susceptibility of mutant plants lacking a functional RNAi response to viral infection and, perhaps most strikingly, the expression of a wide assortment of functionally distinct inhibitors of RNAi by numerous different plant viral pathogens (3, 4). Similarly, there is good evidence in support of a key role for RNAi in antiviral innate immunity in nematodes and insects, including fruit flies and mosquitos (5). Insect genes encode two Dicer proteins, Dcr1 and Dcr2, that function in the generation of mature miRNAs from ∼60-nucleotide (nt)-long pre-miRNA hairpin intermediates and in siRNA generation from long dsRNAs, respectively. While Dcr1 is essential for insect viability, Dcr2 is dispensable, thus allowing the experimental demonstration that Dcr2-deficient fruit flies are hypersusceptible to infection by a range of viruses. This heightened susceptibility correlates with the absence of siRNAs derived from the infecting viral genome.
In considering whether mammalian cells are also able to process viral dsRNAs into antiviral siRNAs, it is important to take into account the defining characteristics of such siRNAs in other systems, especially insects, to ensure that viral RNA degradation products are not erroneously described as siRNAs. Key characteristics of viral siRNAs include the following.
Because they most commonly arise from viral dsRNA replication intermediates, viral siRNAs should derive essentially equally from the viral RNA sense and antisense strands (6). In contrast, the prevalence of viral RNA breakdown products will correlate with the level of the viral RNA of origin, e.g., there are far more sense strands than antisense strands expressed in cells infected with plus-strand RNA viruses. (In principle, siRNAs could also arise from RNA stem-loop structures, and this has indeed been observed in plants and insects. However, transcription of long inverted repeats, flanked by single-stranded RNA sequences, in mammalian somatic cells does not result in the production of significant levels of siRNAs [7], most probably because mammalian Dicer functions exclusively as a dsRNA-dependent exonuclease [8].)
Dicer cleavage products, including siRNAs, have a characteristic size of 22 ± 2 nt (2). Therefore, if an antiviral RNAi response occurs, small RNA deep sequencing will reveal a readily detectable, discrete peak of viral origin in this size range. In contrast, small viral RNA breakdown products are of random size.
Authentic viral siRNAs are associated with RISC and can be specifically recovered by immunoprecipitation of RISC using an Ago-specific antibody. In contrast, viral RNA breakdown products are not enriched in RISC.
Finally, in systems where RNAi is functionally important, viral siRNAs accumulate to very high levels (3). Therefore, deep sequencing should identify many thousands of ∼22-nt viral siRNAs per infected cell if a physiologically relevant antiviral RNAi response is indeed occurring.
DO MAMMALIAN CELLS EXPRESS ANTIVIRAL siRNAs?
Somatic mammalian cells express a single, full-length Dicer protein, here termed DcrS, that plays a critical role in miRNA biogenesis by catalyzing the excision of the miRNA duplex intermediate from the ∼60-nt pre-miRNA hairpin intermediate. Can mammalian DcrS also process long dsRNAs into siRNAs, like insect Dcr2, or is it largely restricted to miRNA biogenesis, like insect Dcr1?
A complicating factor in answering this question is that the expression of long dsRNAs in somatic mammalian cells can induce a potent protein-based innate antiviral immune response called the interferon (IFN) response. This results in the activation of several fairly nonspecific effector mechanisms that induce a global inhibition of mRNA translation, as well as destabilization of mRNAs, and that can eventually lead to cell death. Therefore, unlike in insect cells, which lack any equivalent protein-based antiviral immune response, the detection of a specific antiviral response mediated by RNAi, if it indeed occurs, is obscured by the nonspecific antiviral response mediated by IFN that is induced by the same long dsRNA signal. One approach to the detection of antiviral RNAi is therefore to use deep sequencing to determine if viral siRNAs, with the characteristics described above, are produced in virally infected mammalian somatic cells. Although this issue remains controversial, several studies looking at a wide range of virally infected mammalian somatic cells (6, 9, 10) have failed to detect any significant level of viral siRNAs, including for viruses, such as Dengue virus, that clearly do induce siRNA production in infected insects (11), thus suggesting that mammalian DcrS indeed shares the inability of insect Dcr1 to effectively process long dsRNAs into siRNAs. More recently, two studies have reported direct evidence arguing against a role for RNAi in antiviral defense. In particular, Bogerd et al. (6) used genome editing to derive human cell lines lacking an intact dcr gene and hence incapable of making any siRNAs or even miRNAs, and they found that numerous diverse virus species failed to replicate more rapidly in these mutant cells than in the parental cells expressing DcrS. In addition, Backes et al. (10) engineered an RNA virus, vesicular stomatitis virus (VSV), to express a protein, originally derived from a poxvirus, that effectively blocks the function of all miRNAs and siRNAs by causing their dissociation from RISC. They then showed that this virus does not replicate more rapidly than control VSV either in culture or in vivo. Moreover, the same group also observed that VSV, as well as a second RNA virus, Sindbis virus, replicated equivalently in wild-type mouse embryo fibroblasts (MEFs) and in MEFs engineered to lack a functional dcr gene. Together, these papers therefore argue strongly against the hypothesis that viral infection of somatic mammalian cells results in a protective siRNA-mediated innate immune response. So why would this be the case? In fact, biochemical evidence suggests that the full-length Dicer protein (DcrS) expressed in somatic mammalian cells only very inefficiently processes long dsRNAs into siRNAs, though processing of its normal substrate, pre-miRNAs, into miRNAs is efficient (12). This “defect” has been mapped to the amino-terminal helicase domain of DcrS, which selectively attenuates cleavage of long, perfect dsRNAs, but not pre-miRNAs, by DcrS. Consistent with this idea, deletion of the helicase domain of DcrS selectively increased the catalytic efficiency of human Dicer on a long dsRNA substrate ∼65-fold in vitro (12). This observation not only provided a possible biochemical explanation for the lack of siRNAs of viral origin in infected cells but also raised the possibility that alternative isoforms of mammalian Dicer might exist, perhaps lacking some or all of the amino-terminal helicase domain, that could produce active siRNAs from long dsRNAs. If this was indeed the case, then where might this alternative Dicer isoform(s) be expressed?
DETECTION OF siRNAs IN MURINE OOCYTES AND EMBRYONIC STEM CELLS
While I would argue that efforts to identify functional siRNAs in differentiated mammalian somatic cells have so far been unsuccessful, an exception arises in the case of murine oocytes and embryonic stem (ES) cells. In particular, long dsRNAs have been reported by several groups to be processed into siRNAs in mouse oocytes and ES cells, and these siRNAs appeared fully capable of repressing target gene expression (13–15). Strikingly, mice engineered to transcribe a long inverted repeat were able to process the resultant dsRNA into functional siRNAs in oocytes but not in somatic cells (7). More recently, mouse ES cells infected with RNA viruses were found to express siRNAs of viral origin that were loaded into RISC and appeared to partially attenuate virus replication (16). So why would mouse ES cells generate siRNAs from dsRNAs that, in somatic cells, do not function as a Dicer template? A potential mechanistic explanation for this phenomenon has been reported by Flemr et al. (17), who showed that mouse and rat oocytes, but not somatic tissues, express a novel, amino-terminally truncated isoform of Dicer. This Dicer isoform, called DcrO, derives from a novel retrotransposon-derived promoter located in intron 6 of the genomic dcr gene that is only active in undifferentiated cells. As a result, DcrO lacks much of the amino-terminal helicase domain present in full-length DcrS that was previously reported to inhibit long dsRNA processing in vitro (16), and indeed, DcrO, unlike full-length murine DcrS, proved able to efficiently process long dsRNAs into siRNAs. Of note, deletion of this retrotransposon promoter in the mouse germ line not only blocked DcrO expression without affecting DcrS expression but also led to significant developmental defects. While this might suggest that expression of amino-terminally truncated Dicer isoforms comparable to mouse DcrO might be a common attribute of mammalian species, including humans, this retrotransposon promoter is, in fact, unique to the rodent lineage and it remains unclear whether nonrodent species express a truncated Dicer isoform in germ cells and/or during early development or how such Dicer isoforms might arise. It is, however, tempting to suggest that siRNAs might play a uniquely important role in undifferentiated tissues, especially germ cells, in controlling retrotransposon mobility in order to minimize new and random germ line integration events. Of course, in somatic tissues, such novel insertional events would likely to be far less deleterious as they could not enter the host germ line.
ANTIVIRAL RNA INTERFERENCE IN MAMMALS: A HYPOTHESIS
In conclusion, I would suggest that currently available evidence indicates that RNAi is an important antiviral immune response in nonmammalian species, including in insects and plants, but has been largely supplanted by the IFN response, triggered by the same long dsRNA PAMP, in mammals. Of note, while insects express two Dicer proteins that mediate either miRNA or siRNA biogenesis, mammalian somatic cells express only one full-length Dcr isoform, DcrS, that is incapable of producing significant levels of siRNAs, though it is, like insect Dcr1, fully competent for pre-miRNA processing (16, 18) (Table 1). However, in an as-yet-undefined subset of undifferentiated rodent cells, including oocytes and ES cells and possibly including some other types of stem cells (7, 13–19), long dsRNAs can give rise to functional siRNAs, and these appear able to attenuate viral infections (Table 1). This appears to reflect the expression in these cells of a distinct, amino-terminally truncated isoform of Dicer, DcrO, which has acquired the ability to effectively process long dsRNAs into siRNAs due to loss of an inhibitory domain present in the full-length DcrS protein (17). However, DcrS expression in rodents is dependent on a retrotransposon-derived promoter that is only active in these undifferentiated cells and, perhaps crucially, is absent in primate species. Therefore, we currently do not know if human germ and ES cells, and possibly other stem cells such as hematopoietic stem cells, also differ from differentiated somatic cells in their ability to process long dsRNAs into functional siRNAs and, if so, how this ability might have been acquired. A preliminary analysis of published transcriptome sequencing (RNA-seq) data from human ES cells does not provide a clear answer to the question of whether these cells indeed express a specific, truncated Dicer mRNA isoform (E. M. Kennedy and B. R. Cullen, unpublished data). Although obtaining and culturing human ES cells can be difficult, this is nevertheless an important question whose resolution would have potentially important implications for the evolution of antiviral innate immunity.
Expression of short regulatory RNAsa
ACKNOWLEDGMENT
Work in the Cullen laboratory relevant to this article was supported by Public Health Service grant R21-AI113098 from the National Institute of Allergy and Infectious Diseases.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
