It is well established that type 1 IFNs produced early following virus infection play a critical role in the immune defense against most viruses by limiting virus replication and spread (30
). While various structural components of a virus can elicit type 1 IFN production, the most potent viral inducer is dsRNA, particularly in non-professional innate immune cells such as fibroblasts. Although it is recognized that the majority of viral dsRNA is bound by viral proteins, suggesting that little “free” viral dsRNA exists within a cell, the characterization of multiple cellular dsRNA binding proteins predicts that either entire dsRNA molecules, or portions of these molecules, are available for recognition and binding by cellular proteins. At least four cellular proteins recognize viral dsRNA and mediate type 1 IFN production: TLR3, PKR, RIG-I and MDA-5. While these proteins preferentially bind dsRNA based on its cellular localization, viral origin and/or length, the outcome of dsRNA binding is signaling through the adaptors TRIF (TLR3) or IPS-1 (PKR, RIG-I and MDA-5) to activate IRF3. Activated IRF3 can induce ISGs directly, in the absence of IFN production, or co-operate with additional transcription factors such as IRF1, IRF7 and NF-κB to elicit type 1 IFN production.
Although length plays a role in determining which cellular proteins preferentially bind a given dsRNA molecule, the biological significance of dsRNA length is unclear, particularly given that IRF3 activation ensues regardless of the dsRNA binding protein. To investigate this issue, we produced dsRNA of various lengths from the WNv genome, as the antiviral response to WNv involves TLR3, PKR, RIG-I and MDA-5 (31
) and WNv produces stable dsRNA in fibroblasts following infection, thus confirming the biological relevance of dsRNA species from this viral origin. Here, we provide evidence that dsRNA induces a length-dependent antiviral response, with long dsRNA molecules inducing significantly higher levels of ISG, IRF and IFN transcripts than short dsRNA molecules when equal nM amounts were compared. The length dependence was confirmed upon determination of EC50
values in a standard antiviral assay. Previous studies have shown that activation of TLR3 and PKR requires a minimal length of dsRNA, either to span multiple dsRNA binding sites within a single TLR3 molecule (5
) or to elicit dimerization of two PKR molecules (9
). While activation of RIG-I and MDA5 following dsRNA binding does not appear to have similar requirements, it has been established that MDA-5 preferentially binds long dsRNA molecules and poly IC (11
). Accordingly, a simple explanation for the data presented in this manuscript is that MEFs express elevated levels of MDA-5 relative to RIG-I and thus long dsRNA molecules induce a greater biological response. However, untreated MEFs express low to undetectable levels of both MDA-5 and RIG-I, but rapidly up-regulate the expression of both ISGs following virus or dsRNA treatment (34
), similar to what is seen with PKR. Another possible explanation is that MDA5 binds dsRNA with a higher affinity than RIG-I. To date, comprehensive studies to specifically address this possibility have not been performed. An alternative explanation is that longer dsRNA molecules facilitate better multimerization of MDA5 compared to shorter dsRNA lengths with RIG-I. There is a general precedence with cellular signal transduction that multimerization of pathway components leads to enhanced signal transduction and downstream biological activity. Regardless, the focus of the current study was not to determine which dsRNA binding protein(s) mediates an antiviral response, but rather to delineate the biological outcome of differential dsRNA binding, particularly given that all characterized pathways converge on IRF3. Indeed, it is likely that per dsRNA molecule, long dsRNA molecules have an increased capacity to activate multiple dsRNA binding proteins (of the same or different species), thus potentiating the downstream biological response.
Of particular interest, the dsRNA length dependence was exacerbated in the absence of IRF3. When comparing either individual transcript induction or EC50
values for a given length of dsRNA, a significant difference was observed between the ability of short, but not long, dsRNA molecules to induce ISGs, IRFs and IFN, or to protect wild type versus IRF3-/-
MEFs from virus infection. These results confirm that IRF3 is not essential per se
for ISG and IFN production (4
), but suggest that IRF3 plays a more critical role in response to recognition of short dsRNA molecules. Long dsRNA molecules (3000 bp and poly IC) were equally capable of preventing virus replication in the presence and absence of IRF3. These data suggest that either a novel transcription factor with a similar activity to IRF3 exists, or that in the absence of IRF3, another member of the IRF family can compensate for the loss of IRF3. Recent biochemical and structural studies of the IFNβ promoter show that IRF binding is critical for co-operative association of the IFNβ enhanceosome components and IFNβ transcription (35
). While IRF1, IRF5 and IRF7 have been implicated in type 1 IFN production, it is unlikely that these IRFs are responsible for the effects seen in IRF3-/-
MEFs. We failed to detect differences in the basal expression of IRF mRNA species in the various MEFs, and previous studies showed that ectopic expression of IRF1 fails to restore type 1 IFN induction in the absence of IRF3 (4
) and IRF5 activation does not occur in response to treatment with poly IC (37
). Furthermore, IRF7 expression in fibroblasts is dependent on type 1 IFN signaling [ref. (4
) and ] and IRF7 predominantly activates IFNα promoters, while robust IFNβ expression was observed in the absence of IRF3 following treatment with dsRNA or poly IC. Little is known regarding alternative pathways of dsRNA-mediated type 1 IFN induction involving proteins other than IRFs. While NLRs have been shown to bind to dsRNA (38
) and are important in mediating antiviral immune responses (39
), activation of type 1 IFN has not been observed. Furthermore, is possible that pattern recognition receptors capable of binding dsRNA that signal through alternative transcription factors exist. Thus it remains to be elucidated how dsRNA, particularly large species, mediate type 1 IFN production in the absence of IRF3.
A further surprising observation from these data is the ability of dsRNA to control virus replication in the absence of IPS-1, IRF3 and type 1 IFN production. While IRF3-dependent, IFN-independent and IRF3-independent, IFN-dependent antiviral responses have been observed, to our knowledge this is the first observation of antiviral activity mediated by dsRNA in fibroblasts under conditions where we fail to detect induction of IRFs, IFNs or ISGs. Furthermore, we failed to detect induction of cytokines and chemokines under similar conditions. While we were unable to elicit a complete antiviral response in IRF3&9-/- MEFs at the highest concentration of dsRNA permissible within our experimental system (8.5 nM), we observed a reduction in virus replication ranging from approximately 60% to 90%, depending on dsRNA length. Consistent with data generated in IRF3-/- MEFs, the antiviral response was more robust following treatment with long dsRNA molecules. This antiviral response reduced virus replication of both an RNA virus (VSV) and a DNA virus (HSV-1), albeit with different efficiency. While a 6hr treatment with dsRNA was sufficient to significantly inhibit HSV-1 replication, a 24hr treatment was required to significantly inhibit VSV replication. These data highlight the complex nature of virus-host interactions.
A similar reduction in virus replication was made in MEFs deficient for the mitochondria-associated adaptor IPS-1. While previous studies have shown that IPS-1 is important for activation of IRF3 and NFκB and the subsequent induction of IFNβ, we observed that treatment with long dsRNA molecules was able to induce an antiviral response capable of controlling VSV infection. In both IRF3&9-/- and IPS-1-/- MEFs, however, an extended treatment time (24 hours) was required to elicit a biological response. These data suggest that the cellular pathway(s) that function independently of IPS-1, IRF3 and type 1 IFN preferentially respond to long dsRNA molecules and require sufficient stimulation to accumulate and/or activate constituent signaling components. It is not clear at this time whether a novel dsRNA binding protein participates in this antiviral response or whether the known dsRNA proteins signal through alternative pathways to block virus replication. Studies are underway to address this issue and determine the cellular genes that are induced by dsRNA under these conditions. Overall, these finding further characterize the requirement (or lack thereof) of IRF3 and the type 1 IFN system in the innate antiviral immune response following recognition of dsRNA.