Production of type I IFNs is a critical part of the innate immune response to viral pathogens. Induction of type I IFNs following virus infection is largely controlled by the innate PRRs, which are proteins expressed in cells of the innate immune system. LCMV infection in the mouse, a commonly used model for studying host responses to viruses, has been extensively characterized. However, the molecular mechanisms underlying how LCMV infection induces type I IFNs are poorly defined. In the present study, we have examined the role of LCMV RNA and NP in the activation of the type I IFN response. We demonstrated that LCMV RNA is capable of activating the type I IFN response. Moreover, both dsRNA and 5′-triphosphated ssRNA derived from LCMV genomic RNA are responsible for type I IFN production. Second, the RIG-I/MDA5-MAVS-mediated signaling pathway is essential for the LCMV RNA-induced type I IFN responses both in vitro and in vivo. We provided evidence that LCMV RNA is physically associated with both MDA5 and RIG-I. Third, using bone marrow-derived DCs, we demonstrated that both pDCs and cDCs are able to produce type I IFNs in response to LCMV RNA. Finally, we demonstrated that LCMV NP blocks type I IFN induction.
In the present study, we characterized the innate immune recognition mechanisms involved in detecting LCMV RNA and triggering the type I IFN response. In LCMV-infected cells, LCMV RNA is associated with both MDA5 and RIG-I (Fig. ). This association could account for the activation of type I IFN responses. Rehwinkel et al. recently demonstrated that RNAs bearing 5′-PPPs derived from single-stranded RNA viruses, like influenza virus, trigger type I IFN induction through association with RIG-I (51a
The cellular source of type I IFNs during LCMV infection is not clear (7
). Multiple types of cells might be involved in LCMV-induced type I IFN responses, including spleen marginal zone (MZ) macrophages (34
) and DCs (cDCs and pDCs) (7
). Depletion of MZ macrophages with clodronate liposomes prior to infection with LCMV in the mouse resulted in dramatic loss of type I IFN production (34
). DCs, particularly pDCs, have been demonstrated to play a critical role in producing type I IFN against viral pathogens. Recent studies have also demonstrated that LCMV infection rapidly induces the activation of DCs in the spleen, and this activation is dependent on type I IFN signaling, suggesting a role for both cDCs and pDCs in type I IFN production during LCMV infection. To determine whether both pDCs and cDCs are capable of producing type I IFNs in response to LCMV, we took advantage of bone marrow-derived DCs.
It has been demonstrated that cDCs and pDCs utilize distinctive mechanisms to detect viral RNA and to generate type I IFN production. cDCs and other types of cells predominantly use the MAVS-mediated signaling pathway to generate type I IFNs (23
). In contrast, expression of TLR7 and TLR9 in pDCs is responsible for ssRNA- and unmethylated CpG motif-induced type I IFN production, respectively (26
). In the present study, we demonstrated that LCMV RNA-induced type I IFNs in cDCs are dependent on the MAVS signaling pathway. This is consistent with previous studies. The signaling pathway operating in pDCs responsible for LCMV RNA-induced type I IFN production remains undefined.
The TLR family plays a critical role in regulating the type I IFN response as well as other inflammatory cytokine and chemokine responses to viral pathogens. We have previously observed that TLR2 KO mice had a defective type I IFN response during an acute LCMV infection. The mechanism has not been defined. In the present study, we demonstrated that following LCMV infection, wild-type mice had a greater increase in the mRNA levels of genes involved in type I IFN production, including IFN-α (IFN-α4, non-IFN-α4), IFN-β, and MDA5, than did TLR2 knockout mice (data not shown). Given that the expression of MDA5 and RIG-I is type I IFN inducible, it is conceivable that TLR2 and other unknown molecules could regulate the expression of MDA5 and RIG-I in certain types of cells through either activation of NF-κB signals or other unidentified mechanisms. Of note, the role of TLR2 in virus-induced type I IFN production in certain types of cells has also recently been observed by another group (4
The MDA5/RIG-I-MAVS signaling pathway in vivo
is critical for the LCMV-induced late phase (day 2 p.i.) of type I IFN responses, while the MDA5/RIG-I-MAVS signaling pathway is dispensable for the LCMV-induced early wave (day 1 p.i.) of type I IFN responses. Mice deficient in either MDA5 or MAVS generated comparable levels of type I IFN in the sera at day 1 p.i., while the type I IFN responses in these mice collapsed on day 2 p.i. It has been reported that MDA5 KO mice are capable of generating type I IFN responses against a number of viruses, including influenza virus (NS1 mutant), SeV, Newcastle disease virus (NDV), and Japanese encephalitis virus (JEV) (25
). MAVS KO mice have also been demonstrated to be able to produce levels of type I IFN comparable to those of wild-type mice in response to VSV, although these mice are sensitive to VSV infection (57
). Interestingly, a recent report demonstrated that in response to SeV infection, while MDA5 KO mice generated comparable levels of type I IFN mRNA at day 2 p.i., the levels of type I IFN at day 5 p.i. were dramatically decreased in MDA5 KO mice (16
Like other viruses, LCMV has also evolved countermechanisms to modulate PRRs and other innate signaling molecules to block type I IFN induction. It has been demonstrated that LCMV NP blocks the type I IFN response by targeting IRF3 (39
). IRF3 and IRF7 are the common downstream transcriptional factors involved in both the TLR- and RLR-mediated signaling pathways, and IRF3 has been demonstrated to be a potent transcription factor for activation of the IFN-β promoter (19
). In the present study, we confirmed and extended these findings by demonstrating that LCMV NP blocks type I IFN production through targeting both the MDA5- and RIG-I mediated signaling pathways.
Our experiments demonstrate that LCMV NP physically associates with both RIG-I and MDA5 (Fig. ). It is not clear whether this association is responsible for the inhibitory effect of NP on the activation of type I IFN responses. The fact that mutant NPs that do not suppress the IFN response still associated with RIG-I and MDA5 indicates that simple association does not lead directly to inhibition. It is possible that the site of association is important or that the interaction of the two proteins leads to changes in the tertiary structure of these proteins or their interactions with additional proteins in the pathway. Other possible mechanisms responsible for the inhibitory effect of NP might include the ability of LCMV NP to bind and sequester viral RNA (14
), to degrade the host signaling molecules involved in detection of viral RNA (12
), or to inhibit the positive regulatory loop of the RIG-I/MAVS signaling pathway (13
). Our data do suggest that these two residues (D382 and G385) in LCMV NP are involved in the inhibitory effect of NP but are not required for the interaction with either RIG-I or MDA5.
The fact that LCMV NP-expressing cells are still able to respond to poly(I·C) suggests that different ligands may interact with the RIG-I/MDA5 pathways in different ways. Further experiments involving more specific localization of binding and analysis of multiple protein complexes may be necessary to define the mechanism of the inhibitory action of NP. Interestingly, Lee et al. have recently observed a similar phenomenon. They noted that mice chronically infected with LCMV had defective type I IFN responses to SeV but were capable of producing type I IFNs in response to other stimuli, including poly(I·C), albeit in a reduced manner compared to that of uninfected mice (31
In summary, our current studies define a mechanism by which LCMV infection regulates the type I IFN response. Our current understanding of the recognition and regulation of the type I IFN response to LCMV indicates that this response is dependent upon IRF7 but independent of IRF3. There is no role for the RNA-sensing TLRs (neither TLR3 nor TLR7 seems to be important in the response to LCMV). While the “second wave” (day 2 p.i.) of the IFN response to LCMV is dependent on both MAVS and MDA5, the earliest response (day 1 p.i.) is independent of the RIG-I/MDA5 MAVS pathway, and both are independent of the endosomal TLRs (TLR3, -7, and -9). Our current model indicates that expression of the LCMV NP turns off the induction of type I IFN that is initiated by LCMV RNA interaction with RIG-I and MDA5 (Fig. ).
FIG. 7. LCMV genomic RNA and NP differentially regulate type I IFN responses. LCMV-derived dsRNA and/or 5′-PPP structure activates both the RIG-I- and MDA5-dependent type I IFN responses, whereas LCMV NP inhibits LCMV RNA-induced type I IFN responses (more ...)
We believe that there are dynamic interactions between LCMV RNA, LCMV NP, and the host type I IFN modulators, including MDA5 and RIG-I. These interactions could determine the pattern of type I IFN responses as well as viral pathogenesis during LCMV infection. These results will aid in understanding LCMV viral pathogenesis and the development of new strategies to treat human arenavirus infections as well as other viral diseases.