Special Feature: Rnai And Innate Immunity

Immunology and Cell Biology (2005) 83, 211–216; doi:10.1111/j.1440-1711.2005.01331.x

Immune responses to dsRNA: Implications for gene silencing technologies

Adam J Karpala1, Tim J Doran1 and Andrew GD Bean1

1CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia

Correspondence: Adam J Karpala, CSIRO Livestock Industries, Australian Animal Health Laboratory, Private Bag 24, Geelong, Vic. 3220, Australia. Email: adam.karpala@csiro.au

Received 9 December 2004; Accepted 1 February 2005.



Nucleic acid-induced gene silencing, such as RNA interference (RNAi), induces a multitude of responses in addition to the knockdown of a gene. This is best understood in the context of the antiviral immune response, from which the processes of RNAi are thought to be derived. Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen–host interactions. One aspect of these interactions, the nucleic acid-based immune response, is key to the successful resolution of a viral challenge. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to induction, activation and regulation of antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the activation of dicer-related antiviral pathways, induction of type 1 IFN, and stimulation of dsRNA-activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as toll-like receptor 3. These diverse dsRNA-induced antiviral responses have implications for biotechnologies that use dsRNA to harness one arm of the host antiviral machinery for silencing a specific target gene. The interlinked nature of these response elements means that it may be difficult to completely isolate one element from the other arms of the antiviral response program of an organism. Thus, it is beneficial to understand all aspects of the immune response to dsRNA in order to manipulate these systems and minimize unwanted non-specific effects.


dsRNA, antiviral pathway, RNA interference (RNAi), interferon (IFN), dsRNA-activated protein kinase (PKR)



A crucial instigator of the host response to viral infection is foreign nucleic acid, and in particular, dsRNA. dsRNA triggers a number of antiviral responses that alter the program of a cell (Figure 1). This has implications for RNA silencing technologies that use dsRNA to mediate gene silencing, where the aim is narrowly focused towards a very precise cellular effect. Briefly, RNA silencing, or RNA interference (RNAi), uses dsRNA to exploit a recently discovered intracellular pathway to induce the knockdown or silencing of a specific gene1. Evidence suggests this pathway also constitutes an arm of the antiviral network of eukaryotes that specifically degrades aberrant dsRNA and cognate mRNA2, 3. The wider network of cellular antiviral strategies associated with dsRNA provides a contextual background in which dsRNA-induced gene interference, like RNAi, must operate.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

dsRNA can mobilize several intracellular and extracellular antiviral mechanisms that have broad ranging cellular effects. Once activated, two distinct mechanisms, dsRNA-associated protein kinase (PKR) and oligoadenylate synthetase (OAS), function to shut down protein translation. In addition, gene silencing mechanisms, such as RNA interference (RNAi), leads to the degradation of complementary mRNA when dsRNA activates this pathway. and further, induction of IFN by dsRNA leads to the upregulation of the IFN-stimulated genes (ISG). These genes further promote translational inhibition by increasing proteins such as PKR and OAS, as well as generating an inflammatory response in the organism. The extracellular receptor toll-like receptor (TLR)3 also binds dsRNA and promotes the ISG.

Full figure and legend (14K)


dsRNA origin

Although small dsRNA molecules – termed microRNA (miRNA) – have been isolated from eukaryotic cells and associated with early development4, 5, dsRNA often rings alarm bells for mature vertebrate cells. Foreign dsRNA is a common viral signature linked to the viral replication cycle6. It may be derived from the viral genome (in the case of dsRNA viruses), produced as a replicative intermediate, or produced as an overlapping bi-directional transcript during the replication stage of infection6, 7, 8. Similarly, foreign dsRNA may be introduced to an organism intentionally, as with RNAi, which to some extent, mimics viral infection3 (Figure 2). Principal intracellular antiviral mechanisms that function in vertebrates are known to involve the interaction of dsRNA9, 10, 11. These protective responses are rapid and influence survival, which may help to explain why cells are so responsive to dsRNA12. One branch of the dsRNA-associated antiviral network may be aimed at the viral genome itself.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Intracellular dsRNA can be derived from several sources. dsRNA is a common by-product of viral infection (A) and may result directly from the virus or be produced through transcriptional processes. In addition, dsRNA can be introduced by transfection (B) when gene silencing experiments, such as RNAi, are conducted. This leads to the induction of cellular processes such as cleavage by dicer, and transcriptional activation, such as the IFN-stimulated genes (ISG) that are transcribed as IFN regulatory factors (IRF), becomes active as a result of dsRNA.

Full figure and legend (20K)


dsRNA and gene silencing

Dicer is an endoribonuclease activated by dsRNA13. It is involved in RNA silencing, which specifically degrades the RNA that is complementary to the initiating dsRNA3, 14, 15. The general silencing mechanism is triggered when dsRNA interacts with the dicer protein, which cleaves the aberrant dsRNA into 21 basepair (bp) fragments16. These small interfering RNA (siRNA) are incorporated into an RNA-induced silencing complex (RISC) that then unwinds the duplex siRNA and anneals to the complementary RNA target, subsequently cleaving and destroying it15 (Figure 3). What is the function of a dsRNA-induced gene silencing system? Recent studies suggest that cellular development may, to some extent, be controlled by switching off genes in an RNAi dependent manner4, 17. In addition, mounting evidence supports the idea that RNA silencing is a highly evolved antiviral pathway3. To begin with, it is a highly efficient response to the common viral signature molecule dsRNA and has adaptive advantages in that it impedes a wide range of viruses in plants18. Second, some gene function studies that make use of the RNA silencing pathway in vertebrates display broad antiviral patterns illustrated by the induction of IFN and dsRNA-activated protein kinase (PKR)19. Thus, when these hosts are treated with dsRNA, they appear to be responding in an antiviral-like manner to the introduced dsRNA (as though it were a viral signature). In addition, there are several viral countermeasures that are directed at evading this host defence, some targeting the dsRNA aspect directly20. Both plants and animals produce a number of these viral suppressors, adding support to the evolved antiviral nature of RNA silencing3, 21, 22. Furthermore, plants that have defective RNA silencing pathways are susceptible to certain viruses22. Since its discovery in animal systems, the dsRNA-activated arm of antiviral immunity that degrades a specific gene has been successfully exploited with the use of RNAi.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

A generally accepted model of the RNA interference (RNAi) mechanism. Long dsRNA is introduced to a cell. Once it is in the cytoplasm, the dicer enzyme cleaves the dsRNA into 21–23 bp fragments (small interfering RNA; siRNA) with 2 nucleotide 3' overhangs. Following cleavage, RNA-induced silencing complex (RISC) loads and unwinds the siRNA, and binds to the complementary target mRNA which is subsequently cleaved by the RISC. Following cleavage, the RISC complex disassembles and is ready to load another siRNA for cleavage of additional mRNA.

Full figure and legend (19K)


Manipulating the antiviral response by dsRNA-induced RNAi

RNAi has enabled great leaps forward in the area of gene function studies and offered new hope to therapeutics that might benefit from gene knockdown. For example, in Caenorhabditis elegans, approximately 16 700 genes have been screened for functionality using RNAi23. Furthermore, viruses causing diseases such as AIDS and hepatitis have been targeted with some success using various RNAi techniques24, 25. Nonetheless, these advances, like any new biotechnology, have associated difficulties in vertebrates. Intrinsically, many difficulties are connected with the use of dsRNA. The complex antiviral pathways associated with dsRNA, some outlined here, are likely to be provoked upon the introduction of dsRNA molecules into these organisms. Initially, research conducted by Elbashir et al. (2001) demonstrated that siRNA, in contrast to long dsRNA, did not induce non-specific antiviral pathways in mammalian systems, and consequently RNA silencing techniques advanced rapidly19. It appeared that short dsRNA molecules that are less than 30 bp escaped the antiviral defence net. More recent research, however, challenges some of these notions. Sledz et al. (2003)26 demonstrated that the IFN-mediated JAK-STAT pathways11 and PKR are induced upon introduction of siRNA10. Further studies by Pebernard and Iggo (2004) shows that vectors expressing short hairpin RNA (shRNA) constructs using particular promoter sequences can induce the antiviral intermediary oligoadenylate synthetase (OAS)27. The dsRNA interactions that instigate these antiviral phenomena may be diverse and widespread because there are several intracellular dsRNA binding proteins potentially capable of mediating antiviral effects28. Some studies show that siRNA stimulate toll-like receptor (TLR)3 and provoke an antiviral response29, while others implicate PKR as the link to antiviral induced pathways11. Thus, antiviral mechanisms associated with RNAi may be widespread and need careful consideration. In addition, dsRNA is a potent inducer of IFN in vertebrates30, 31, requiring as little as a few intracellular molecules to invoke a response32.


IFN protects against viral infection

One of the most significant and well understood early antiviral responses is the activation of the IFN pathway9. IFN are a group of cytokines comprising both type 1 and 2 IFN as well as IL-28 and -29. Only the type 1 IFN, which include several IFN-alpha and a single IFN-beta type, are inducible by virus directly9. Briefly, IFN-alpha and -beta interfere with viral replication by modulating PKR and OAS, which restricts the translation machinery of the host as well as produces numerous downstream effects important for the host control of virus10, 33. OAS itself is activated by dsRNA and is partly responsible for obstructing cellular transcription by alteration of ATP to 2'–5' linked oligomers34. These products bind and activate the endoribonuclease RNaseL, which begins destroying diverse RNA such as ribosomal RNA, further inhibiting protein synthesis,35, 36 and viral RNA to clear it34. In addition to the translational inhibition mechanisms, IFN induce genes that can lead to apoptosis, another important host viral control feature. Paracrine effects include the increase of MHC class I, which helps NK cells distinguish their viral infected targets, and signals that activate the antiviral response of neighbouring cells, impeding the progression of viral infection9. The central importance of IFN-related antiviral responses is demonstrated by increased virulence when viruses produce inhibitors that target IFN36 and additionally, IFN-beta mouse knockouts that become susceptible to numerous viruses33. Although IFN can be triggered by several host pathogen interactions, it is often triggered by dsRNA in the context of viral infection12, 30, 37. Therefore, dsRNA interactions can be decisive in determining the outcome of viral invasion. Consequently, strategies that endeavour to manipulate cellular processes by using dsRNA need to take into account the potential for a wider impact on the organism. A key to understanding dsRNA-induced antiviral mechanisms would not be complete without discussing the pivotal dsRNA mediator PKR, which has a fundamental role in the antiviral network38. Several of the dsRNA-induced mechanisms rely on this protein.


PKR: Intracellular dsRNA recognition factor

It is known that PKR, a cytoplasmic dsRNA interactive protein, can bind dsRNA and influence the regulation of IFN-alpha and beta, and the associated downstream effects, upon dsRNA stimulation38, 39, 40. However, it is not clear whether PKR is the crucial dsRNA intracellular recognition protein that initiates the generation of IFN. There may be additional intracellular dsRNA recognition proteins or factors that contribute to IFN-alpha and beta being rapidly transcribed upon viral infection or cytoplasmic exposure to dsRNA30, 36. In addition to inducing IFN, active PKR can bring about the shutdown of protein synthesis via the phosphorylation of the initiation factor elf241. This radical arm of the antiviral program is targeted at immediate prevention of the virus commanding the host machinery41. To mediate the phosphorylation of elf2, PKR requires dsRNA as an activation element to facilitate its own autophosphorylation42. PKR is remarkably complex, having a number of protein substrates, and it is known to contain at least two dsRNA binding motifs (dsRBM)28. In the presence of dsRNA greater than 30 bp, both binding sites may be occupied and the activity increases relative to the length of dsRNA up to 85 nucleotides in length43. However, each individual site requires as little as 11–16 bp of dsRNA, around one helical turn of dsRNA, for binding42. When only one of these sites is bound, PKR can be inhibited or activated depending on the site effected28. Furthermore, PKR can be differentially modulated by different species of dsRNA, with as few as 11 bp having an effect42, 43. This is an important consideration in the context of dsRNA-induced gene silencing, where it may be assumed44 that dsRNA smaller than 30 bp escapes activation of the dsRNA-induced IFN network. Moreover, the sequence and secondary structure of the dsRNA are features that may alter PKR activity28, 43. As a result, a particular virus or introduced dsRNA may yield specific PKR activity depending on the associated nucleic acid features45. Cell type, growth and health status might also effect what role PKR will execute46, and must be considered when RNAi-related data are compared between experiments47. In addition to the intracellular recognition proteins, there are extracellular dsRNA-induced antiviral mechanisms.


dsRNA recognition by TLR3

Exogenous exposure to dsRNA can modulate cellular responses and induce antiviral pathways, particularly IFN30, 31. TLR3, a dsRNA pattern recognition receptor, is found on the surface of many cell types, including fibroblasts and macrophages30 and some dendritic cells48 and, to date, is the only known external dsRNA recognition receptor. When dsRNA interacts with TLR3, it induces IFN-beta as well as IL-6 and -12 and TNF-alpha, which help shape the overall immune response30, 31. It has been suggested that PKR and TLR3 may function as separate intracellular and extracellular viral detection mechanisms, respectively49, thus enabling an external immune surveillance mechanism. The external recognition of dsRNA is likely to be important for uninfected cells as an early warning system of imminent viral infection when dsRNA is released from lysed cells9. This scenario is mimicked when dsRNA is exogenously delivered in RNAi experiments. Kariko (2004) showed that TLR3 does mediate IFN effects in siRNA-induced gene silencing experiments29. TLR3 has further been identified in the cytoplasm of immature dendritic cells and shown to be dsRNA responsive50, which could impact on plasmid-delivered shRNA molecules that bypass external recognition. If the precise effects that are invoked by a nucleic acid are understood, they may be used to an advantage. For example, the reduction of viral load in a mammalian host using RNAi could be facilitated by the wider non-specific antiviral responses. In this case, delivered dsRNA could perform the role of gene silencing as well as having a therapeutic-like effect – a potential advantage in a disease model using RNAi. In addition to the wider antiviral gene expression patterns associated with the use of RNAi, modulation of gene expression may arise that is unrelated to antiviral mechanisms. These should be identified separately as they relate to design aspects of RNAi in contrast to the antiviral responses of a cell.


Gene silencing off-target effects

Complications occur with RNAi when a gene closely related to the target gene is destroyed by the specific siRNA – this is known as an 'off-target' effect51, 52. Some microarray studies show that one or even 2 bp mismatches between an siRNA and an unintended target is enough to impede that gene51. Reduction of gene expression as a result of these off-target effects may result in outcomes that may appear to be related to changes in the antiviral pathways. This can be avoided by the careful choice of gene targets and the selection of siRNA47. In addition, the translation of an mRNA could be inhibited when an siRNA contains partial sequence similarity to the mRNA and consequently binds and blocks it52. This concept was borne out in a study that targeted a gene using 10 different siRNA52. While the expression of the target gene was reduced, changes in the expression of two unrelated proteins were observed and shown not to be antiviral related52. It was speculated that an miRNA-like inhibition of translation had occurred where some sequence mismatches are tolerated and binding of the target RNA by the siRNA blocks translation processes4, 52. In the context of therapeutics, off-target effects are of great concern because of the potentially catastrophic effects such as the targeting of a gene necessary for survival. It is therefore vital to understand all of the implications of RNAi, particularly the possibility of multiple dsRNA-induced gene silencing responses.


Multiple dsRNA-induced RNAi pathways

Recently, another RNA silencing mechanism was identified in vertebrates: switching off the transcription of genes by DNA methylation53. Research demonstrated that introduced 21–25 bp long siRNA mediators specifically induced the methylation of DNA and the binding of histones, which led to repression of E-cadherin gene expression53. The RNA silencing occurred before transcription events, contrasting with the post-transcriptional RNAi processes described previously. As DNA methylation was induced using longer siRNA molecules up to 25 nucleotides in length, it is likely that there are several dicer enzymes involved in the different RNA silencing pathways. If multiple RNA silencing pathways are maintained in a cell, it will be interesting to discover when one silencing mechanism is favoured over another. RNA silencing mechanisms have a fundamental dependence on dsRNA interactive proteins, the processes of which are still being demystified. There are other cellular functions that depend on the family of dsRNA binding proteins and are less understood in the context of RNA silencing.


Other dsRNA inducible responses

Many intracellular proteins are capable of binding dsRNA28 and these may have unexpected implications for RNA silencing. Table 1 summarizes several identified dsRNA binding proteins. Some of their functions are known; however, others are not fully characterized and as such their full impact on RNAi may not be known. That the complete array of dsRNA binding proteins may impinge on RNAi experiments is highlighted in research conducted with antisense silencing54. In this case, it was shown that antisense targeted at a specific gene was rarely able to reach the intended target due to the presence of the RNA binding proteins54. This process, known as an aptamer effect, was further related to the formation of new products that could induce downstream effects in an organism54. Taking this into account, in addition to the wide array of antiviral mechanisms of the cell, it is reasonable to suggest that the cell's array of dsRNA binding proteins could induce a variety of cellular responses when exposed to dsRNA.



The antiviral mechanisms induced by dsRNA are vital to protecting the host. These diverse and complex responses appear to be orchestrated in such a way as to generate a comprehensive antiviral strategy. RNAi directed gene silencing aims to use one arm of the antiviral response in isolation from all the other cellular events. If this is to be effectively achieved, then it is necessary to have a good understanding of how the various elements of the antiviral responses are orchestrated. A key to greater understanding of the complex antiviral response is very dependent on the dsRNA interactions with the cellular machinery. This viral signature can alter the intracellular program of an organism through the induction of several antiviral mechanisms such as dicer related pathways, IFN and PKR. There are, however, some uncertainties, including how dsRNA is initially recognized and what features of this nucleic acid are important for driving the many branches of the antiviral system. In addition, there may be unidentified dsRNA interactions with the numerous other intracellular dsRNA recognition proteins28 that effect gene silencing outcomes. Finally, multiple RNAi pathways implies that a consideration of the individual mechanisms is required in order to harness a specific effect. These issues will be important to consider for nucleic acid-based techniques, such as RNAi, which are likely to play a valuable role in the future of antiviral therapeutics.



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This work is supported in part by the Australian Poultry Cooperative Research Centre (CRC).


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