As discussed above, the TLRs play an important role in sensing viral PAMPS that are present within the extracellular compartment, as well as in endosomes. In certain contexts, TLRs can receive viral nucleic acids generated from viruses that replicate in the cytoplasm, via an autophagy mechanism. A role for intracellular sensors in the clearance of viruses that replicate and reside within the cytosol of cells has recently emerged. Following the generation of mice lacking TLRs and examination of their susceptibility to virus infections, it became clear that additional sensing mechanisms must also exist and contribute to anti-viral defenses. The last decade or more has revealed numerous additional classes of innate sensors. Of particular relevance to anti-viral defenses was the discovery of specialized classes of cytosolic nucleic acid sensors, termed RIG-I like receptors (RLRs), which recognize intracellular RNA that is introduced to the cytosol during viral infection or that accumulates during replication. Additionally, a diverse selection of intracellular DNA sensors which recognize viral DNA within the cytosol have also emerged.
3.1. The RIG-I like Receptor Family
The RLR family is comprised of three DExD/H box RNA helicases: retinoic acid-inducible gene (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and laboratory of genetics and physiology-2 (LGP-2) [60–64]. Both RIG-I and MDA-5 are comprised of tandem N-terminal caspase activation and recruitment domains (CARDs) followed by a DExD/H box RNA helicase domain which has ATPase activity and a C-terminal repressor domain (RD). Unlike RIG-I and MDA-5, LGP-2 lacks the N-terminal CARD domains, containing only the RNA helicase domain. As such, LGP-2 was postulated to act as a negative regulator of the other RLRs [61,63]. Under resting conditions, RIG-I resides in the cytoplasm in an inactive form that is auto inhibited by its regulatory domain. Upon viral infection, RIG-I undergoes a conformational change by which it dimerizes in an ATP dependent manner . The activated multimeric form of RIG-I or MDA5 then interacts with the downstream adaptor protein mitochondrial antiviral signaling protein (MAVS), also known as VISA, IPS-1, and CARDIF, via CARD-CARD interactions. MAVS is localized to the outer leaflet of the mitochondrial membrane, which is an essential location to support downstream signaling. Recently, MAVS was also shown to be localized on peroxisomes, from where it induces an early antiviral response through the direct induction of a subset of anti-viral genes via the transcription factor IRF1. Upon engagement of RIG-I or MDA5 with MAVS, MAVS activates the IKK-related kinase, TBK1/IKKi, which activates IRF3/IRF7, resulting in the transcription of type I interferons. MAVS also activates NF-κB through recruitment of TRADD, FADD, caspase-8, and caspase-10 [65–69].
3.2. RNA Recognition by RLRs
The RLRs are critical components of the anti-viral defense pathway in many cell types including fibroblasts, epithelial cells, and conventional dendritic cells . Initially, it was thought that both RIG-I and MDA-5 recognized the synthetic dsRNA, polyinosinic acid (polyI:C). However, studies from RIG-I and MDA-5 deficient mice determined that MDA-5 alone was responsible for interferon production by polyI:C stimulation . Instead, RIG-I recognizes 5′-triphosphorylated, uncapped ssRNA, which is a common feature in many viral genomes. However, it is unable to recognize the capped 5′-ppp ssRNA from the host cell [72–74]. These finding suggest that RIG-I uses the 5′ end of a transcript to discriminate between viral and host RNA. MDA-5 distinguishes between viral and host RNA not by its 5′ end, but rather by the length of the RNA sequence; long dsRNA is not naturally present in host cells and acts as a ligand of MDA-5. In addition to recognizing 5′-triphosphate RNA, RIG-I is also capable of recognizing short dsRNA, which is produced as a byproduct of viral replication .
RIG-I and MDA-5 appear to differentially recognize different classes of RNA viruses. Studies involving RIG-I deficient mice implicated RIG-I in the recognition of vesicular stomatitis virus (VSV), rabies virus, SV, Newcastle disease virus (NDV), RSV, measles virus, Influenza A and B, hepatitis C virus (HCV), Japanese encephalitis virus, and ebola virus [53
,70,71,76–78]. Studies from MDA-5 deficient mice show that MDA-5 is able to recognize EMCV, theiler’s virus, and mengo virus [71,77]. All of these viruses do not contain a 5′ triphosphate RNA, but are able to produce long dsRNA, providing further evidence that MDA5 discriminates between self and non-self RNA based on sequence length and not the 5′triphosphate. More recently studies have shown that both CVB and poliovirus are dependent on MDA-5 for type I IFN production [79,80]. Moreover, some viruses, such as dengue, West Nile virus, and reovirus, signal through a combination of both RIG-I and MDA-5 [79,81,82].
As discussed above, LGP-2 lacks N-terminal CARD domains, and was first thought to be a negative regulator of RLR function [61,63]. Initial studies found that overexpression of LGP-2 decreased the capacity of SV and NDV to induce interferon production. Evidence that LGP-2 could associate with RIG-I through mutual RD domains led to the proposal that LGP-2 directly prevented RIG-I association and activation. Consistent with this idea, interferon signaling was found to be increased in LGP-2 deficient mice responding to polyI:C, providing evidence for negative regulation of MDA-5 as well . A second in vivo study using LGP-2 deficient mice as well as mice harboring an inactive ATPase in the DExD/H-box RNA helicase domain showed that LGP-2 acted as a positive regulator of RIG-I and MDA-5-mediated signaling after infection by RIG-I and MDA-5-specific RNA viruses. This phenotype is consistent with the possibility that LGP-2 might promote RNA accessibility, thus enabling RIG-I or MDA-5 dependent viral recognition. Further studies on these mice will no doubt clarify this upstream mechanism and the role of LGP-2 in this pathway.
Another member of the DExD/H box RNA helicase family, DDX3, has also recently been implicated in anti-viral defenses. Schroder et al. found that the vaccinia virus protein K7 inhibited IFNβ induction by binding to DDX3, which led to the discovery that DDX3 had a positive role in the RLR signaling pathway . A more recent study reported that DDX3 binds to both polyI:C and viral RNA introduced into the cytosol and associates with MAVS/IPS-1 to upregulate IFNβ production. These results led the authors to speculate that DDX3 might enhance RNA recognition, forming a complex with RIG-I and MAVS to induce interferon production . Further studies are required to determine whether DDX3 is a bona fide RNA sensor or a component of the RLR signaling pathway in order to fully understand the function DDX3 plays in anti-viral surveillance and signaling.
3.4. Cytosolic DNA Sensors
Prior to the discovery of TLR9, it was known that DNA derived from pathogens could activate fibroblasts to produce type I IFNs . This phenomenon was ignored or underestimated for decades and was rediscovered following the observation that transfection of pathogen-derived dsDNA activated a TLR9 negative thyroid cell line to upregulate various immunological genes . Akira and colleagues subsequently demonstrated that TLR9−/− MEFs, which failed to respond to CpG DNA, produced large amounts of IFN in response to transfection with synthetic b-form dsDNA or genomic DNA isolated from bacteria, viruses, and mammalian cells . This was similar to findings presented by the Medzhitov lab using a 45 bp dsDNA region from the Listeria monocytogenes genome. Cytosolic administration of dsDNA did not appear to utilize any known TLRs to induce interferon since cells from mice lacking both MyD88 and TRIF responded normally.
Like the cytosolic RNA recognition pathways, cytosolic DNA recognition also leads ultimately to activation of TBK1 and IRF-3 and production of type I IFNs. However, the signaling pathway linking upstream DNA sensors to TBK1 are poorly characterized. TBK1 associates with DDX3, a DEAD box RNA helicase, which regulates IFNβ transcription via IRF-3 [84,85]. In addition, TBK1 interacts with the exocyst protein Sec5 in a complex that includes the recently identified endoplasmic reticulum (ER) adaptor stimulator of interferon genes (STING) [69,88–90]. STING plays a central role in the signaling pathway upstream of TBK1 following HSV infection . STING also interacts with the ER translocon components Sec61β and TrapB in a manner essential for regulation of cytosolic DNA-induced type I IFN production, although the mechanistic understanding of this finding is not known . In unstimulated cells, STING localizes to the ER and perhaps ER-associated mitochondria . Following stimulation with cytosolic DNA and HSV-1, STING translocates to perinuclear foci, via the Golgi . STING localizes partially to endosomes, particularly Sec5 positive structures , whilst another report has demonstrated that STING localizes to vesicular structures, which are not peroxisomes, mitochondria, endosomes or autophagosomes . Further work is required to clarify the precise subcellular localization of STING. What is clear is the essential role of STING in cytosolic DNA sensing pathways. Much less clear is the mechanisms or receptors which act upstream of STING. A growing number of DNA sensors have now been implicated and will be outlined below.
DNA-dependent activator of IFN-regulatory factors (DAI) was among the first of the cytosolic DNA sensors to be discovered. It is composed of two binding domains for left-handed, Z form DNA, although the protein can recognize B form DNA as well. When DAI was exogenously expressed in L929 cells, it increased type I IFN production in a dose dependent manner following stimulation by both B and Z form DNA. Similarly, knockdown of DAI with siRNA impaired type I IFN production in response to DNA, the 45 bp interferon stimulatory DNA (ISD) from Listeria and the herpesvirus, HSV-1 [92,93]. The production of type-1 interferons by fibroblasts in response to HCMV was also found to be dependent on DAI . DAI-knockout mice were subsequently generated, and surprisingly, cells derived from DAI deficient mice respond normally to synthetic and viral dsDNA [92,95]. These results suggested that DAI might play a cell type specific, and redundant role in sensing cytoplasmic DNA, and that other sensors must also be necessary for inducing these responses.
3.6. RNA Pol III
As discussed above, both synthetic and viral RNA trigger the production of type I IFNs via RIG-I. Although, the RLRs are sensors of RNA, some data has suggested a role for this system in detection of DNA. A somewhat surprising finding was that synthetic B-form dsDNA can also induce IFNβ production in human cells in a manner that was dependent on the RIG-I adapter molecule MAVS [52
]. These findings suggested the existence of an unknown DNA sensor that would signal via MAVS. Recently, two independent studies have provided an explanation for these findings and shown that AT-rich DNA can be transcribed by RNA polymerase III into 5′-ppp RNA, which subsequently activates RIG-I [52
]. This pathway was reported to be involved in type I IFN induction during EBV infections where the EBERs are transcribed by RNA polymerase III [56
]. This indirect DNA-sensing system was also reported to be involved in induction of type I IFN following HSV-1 or Legionella
In addition to DAI and RNA Pol III, Leucine-rich repeat flightless-interacting protein 1 (LRRFIP1) has recently been implicated as a regulator of DNA-driven innate immune signaling. LRRFIP1 was found to bind to the drosophila homolog flightless I and play a role in actin organization during drosophila embrogenesis. In a study using Listeria monocytogenes to screen for potential cytosolic DNA sensing molecules, siRNA against LRRFIP1 was found to inhibit type I IFN production induced by the bacteria. The authors showed that the IFN response to VSV was dampened in these cells as well. Furthermore, knockdown of LRRFIP1 inhibited IFN production in response to polyI:C, and the synthetic DNA species, poly(dG:dC) and poly(dA:dT), implicating LRRFIP1 in the recognition of both dsRNA and both B and Z form dsDNA. Surprisingly, this function is independent of RNA Pol III. LRRFIP1 does not regulate IRF3 activation but instead appears to regulate a novel β-catenin-dependent coactivator pathway. LRRFIP1 binds RNA or DNA and leads to phosphorylation of β-Catenin, which subsequently translocates to the nucleus where it associates with the p300 acetyltransferase at the IFNβ1 promoter, leading to increased IFNβ production . Although LRRFIP1 has been implicated in the recognition of both Listeria monocytogenes and VSV, further studies are needed in order to determine its role in sensing other viruses, particularly DNA viruses.
While analyzing immune responses to a dsDNA region derived from the VV and HSV-1 genomes, Bowie et al. identified IFI16 as a DNA binding protein which interacted with these dsDNAs. IFI16 is a member of the PyHIN (pyrin and HIN200 domain-containing) protein family. The PHYIN family consists of 4 family members: IFIX, IFI16, MNDA and AIM2. All contain one or more HIN200 domains, which recognize DNA as well as a pyrin domain. Knockdown of IFI16 or p204 (a member of the murine PYHIN family) led to a reduction in IFNβ responses to these dsDNAs while responses to the RNA virus SV was unaffected. Although IFI16 is primarily nuclear in most cell types, in macrophages IFI16 also localized to the cytosolic compartment where it co-localized with dsDNA introduced via lipofectamine. Association of IFI16 with STING was required for the production of IFNβ in response to these DNA motifs. siRNA knockdown of IFI16, and its mouse homolog p204 led to a decrease in IRF3 and NF-κB activation and IFNβ gene induction following infection of cells with HSV-1 .
3.9. DDX9 and 36
Also in the family of DExD/H box RNA helicases, DHX9 and DHX36 have recently been shown to recognize and bind CpG-B and CpG-A DNA, respectively in plasmacytoid dendritic cells. Activation of DHX9 leads to IRF-7 activation and IFNα production, while activation of DHX36 leads to the activation of NF-κB and the production of IL-6 and TNFα. siRNA knockdown of DHX9 and DHX36 inhibited cytokine production in response to the DNA virus HSV-1, while response to the RNA virus influenza A was unaffected .