In our present study, we sought to identify cellular factors targeted by arenavirus NP to mediate suppression of innate antiviral signaling. The central findings derived from our studies are that (i) LCMV NP prevents activation of IRF3 by blocking phosphorylation of the transcription factor; (ii) LCMV NP specifically targets the IKK-related kinase IKKε but not TBK-1; (iii) the specific binding of NP to IKKε is conserved within the Arenaviridae; (iv) LCMV NP associates with the kinase domain of IKKε involving NP's C-terminal region; and (v) binding of LCMV NP inhibits the kinase activity of IKKε.
Previous studies demonstrated the ability of arenavirus NP to prevent activation and nuclear translocation of IRF3 in response to viral infection (30
). Here we demonstrate that, upon infection with SeV, which activates the RIG-I/MAVS pathway, LCMV NP blocks IRF3 phosphorylation required for dimerization and subsequent nuclear translocation of the transcription factor (17
). Considering the proposed role of the RIG-I/MDA5/MAVS pathway in innate detection of arenaviruses (14
), we probed the interaction of LCMV NP with selected components of the RIG-I/MDA5/MAVS pathway implicated in IRF3 phosphorylation using a co-IP approach. Although by no means comprehensive, our co-IP analysis revealed a strong and specific interaction of LCMV NP with IKKε but not with TBK-1, RIG-I, MAVS, TRAF3, or IRF3 under our experimental conditions. The lack of detectable co-IP of LCMV NP and RIG-I in our hands seems in contradiction to previous reports that demonstrated an interaction of LCMV NP with RIG-I. The reasons for this apparent discrepancy are not clear and may be due to the more stringent washing conditions applied in our co-IP protocol. Of note, the interaction between LCMV NP and RIG-I reported by Zhou et al. was not affected by the D382A mutation that abrogates the ability of NP to suppress type I IFN induction (56
) as well as the high-affinity interaction with IKKε (this study). Examination of NPs derived from representative members of the major arenavirus clades revealed evolutionary conservation of the specific interaction of NP with IKKε but not with TBK-1. Confocal microscopy combined with quantitative image analysis revealed that, in LCMV-infected cells, IKKε strongly colocalized with LCMV NP but not with its cellular binding partner MAVS. This suggests that NP somehow sequesters IKKε and prevents its recruitment to the MAVS signaling platform located on the outer mitochondrial membrane. Whether this sequestration is linked to sites of virus replication remains to be determined. Notably, when associated with NP, IKKε seems catalytically inactive, suggesting as rather unlikely a role of IKKε in viral replication via phosphorylation of cellular proteins or viral proteins or both.
The specific ability of arenavirus NP to target IKKε, but not TBK-1, was unexpected and seems different from the pattern seen with other viruses. Targeting of IRF3 phosphorylation as a viral strategy to subvert induction of type I IFN responses was first demonstrated for the phosphoprotein (P) of rabies virus, which was shown to interfere with phosphorylation of IRF3 by TBK-1 (5
). More recent studies mapped this activity to a specific domain within the P protein that is dispensable for replication (46
). In contrast, we found that the C-terminal region of LCMV NP, which is required for virus RNA replication and transcription, seems involved in binding to IKKε. Notably, the association of NP blocks the catalytic activity of IKKε, as evidenced by a lack of autophosphorylation and the inability of NP-bound IKKε to phosphorylate IRF3 or NP. This is different from the mode of action reported for the paramyxovirus V proteins and Ebola virus VP35, which interfere with both TBK-1- and IKKε-mediated IRF3 phosphorylation by acting as competing pseudosubstrates (25
). Moreover, upon phosphorylation by TBK-1 and IKKε, paramyxovirus V proteins are degraded (25
), whereas LCMV NP remains stable over time. Therefore, regarding specificity and the molecular mechanism of inhibition of IKKε and TBK-1, arenavirus NPs behave differently from IFN antagonist proteins of Rhabdoviridae
, and Filoviridae
Arenavirus NP is a versatile protein that fulfills a plethora of functions in virus replication, virion assembly, and interaction with host cell factors. The examination of a set of previously described and well-characterized NP mutants (29
) revealed a role of the C-terminal domain of NP in binding to IKKε. The C-terminal domain of NP is involved in binding to the viral matrix Z protein (39
) and contains a 3′-5′ exonuclease domain, whose activity was linked to the anti-IFN activity of NP (15
). The interaction with Z and the suppression of IFN activation map to partially overlapping regions of NP but also involve distinct functional domains (39
). Our present study was limited to NP mutations causing impairment in the 3′-5′ exonuclease function and IFN suppression. All mutants tested showed significantly weaker binding to IKKε, suggesting that overlapping domains of NP are involved in 3′-5′ exonuclease function and IKKε binding. Future comprehensive mutation-function studies would be required to structurally separate the two functions and to distinguish their individual contributions to virus-induced suppression of the induction of type I IFN.
The specific ability of arenavirus NP to target IKKε, but not TBK-1, was unexpected, because existing evidence indicates a more important role for TBK-1 in the induction of type I IFNs in response to virus infection (16
). Analysis of murine embryonic fibroblasts derived from mice deficient in IKKε or TBK-1 suggested a predominant role for TBK-1, rather than IKKε, in IFN induction in response to dsRNA and SeV infection (16
). However, in macrophages, the IFN response to SeV was normal in TBK-1-deficient cells (40
), indicating cell-type-specific differences. To address the relative contributions of TBK-1 and IKKε to IRF3 phosphorylation and IFN-β induction in response to SeV in our system, we performed RNAi. In HEK293 cells under our experimental conditions, we found that both TBK-1 and IKKε were required for optimal induction of IFN-β upon SeV infection. The apparently nonredundant roles of IKKε and TBK-1 suggest that SeV-induced IRF3 phosphorylation in HEK293 cells involves a complex requiring both kinases. It is conceivable that NP targeting IKKε may also affect the function of TBK-1, resulting in a block in IRF3 phosphorylation. However, several lines of evidence suggest that TBK-1 can function in the absence of IKKε (16
). Innate detection of RNA viruses, e.g., by Toll-like receptor 3 (TLR-3), results in specific activation of TBK-1 but not IKKε via the signaling adapter TRIF (49
). The absence of a direct interaction between arenavirus NP and TBK-1 found here indicates that arenaviruses may use other strategies to evade TBK-1-dependent pathways. One possible strategy is illustrated by the degradation of viral RNA by the 3′-5′ exonuclease activity of LCMV NP that is linked to its function as a type I IFN antagonist (15
). By degradation of viral RNA, the 3′-5′ exonuclease of NP may eliminate the “danger signal” preventing recognition by RIG-I helicases. The specificity of NP for IKKε, but not TBK-1, would allow persistent arenaviruses to leave major TBK-1-dependent pathways of innate immunity intact. This strategy may contribute to protecting the natural reservoir host against infection by other pathogens, including DNA viruses, bacteria, fungi, and parasites.
The remarkably conserved specificity of arenavirus NPs for IKKε suggests other nonredundant roles for IKKε in arenavirus-host cell interaction. IKKε has also been implicated in activation of NF-κB in response to phorbol myristate acetate (PMA) and T cell receptor activation but not tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) (42
). In breast cancer, IKKε is known to be an oncogene involved in uncontrolled NF-κB activation (3
). Interestingly, as noted in the accompanying article, Rodrigo et al.
have reported that LCMV NP is able to specifically inhibit activation of NF-κB in response to virus infection with only a mild effect on NF-κB induction by TNF-α (46a
). In addition to its role in activation of IRF3 and NF-κB, IKKε but not TBK-1 affects IFN-regulated gene expression by phosphorylation of STAT1 on serine 708 (54
). IKKε-mediated phosphorylation of STAT1 affects the quality of the gene expression profile in response to type I and type II IFNs (37
). Apart from antiviral signaling, IKKε has been implicated in the regulation of energy metabolism in models of obesity (8
) and cell transformation in cancer cells (19
). Since arenaviruses are carried in nature by persistent infection of their reservoir rodent species, a major thrust of our current research is to study the role of the arenavirus NP-IKKε interaction in the context of viral persistence. The specificity of NP for IKKε may allow using the virus as a molecular probe to dissect nonredundant functions of IKKε and to elucidate their role for arenavirus-host interaction.