The IFN-I system serves as a first line of defense against virus infection. Induction of IFN-I during viral infection requires the activation of the transcription factors NF-κB, IRF3, and AP-1 and is essential for the expression of many host genes involved in antiviral defense mechanisms (19
). Accordingly, viruses interfere with these pathways via a variety of mechanisms, including targeting of key cellular transcription factors by viral proteins (19
), which facilitates virus multiplication and pathogenesis.
We have shown that, with the exception of TCRV-NP, all arenavirus NPs tested inhibit nuclear translocation and transcriptional activity of IRF3, supporting a critical role of NP in the inhibition of the host IFN-I response in arenavirus-infected cells (53
). In this report, we provide evidence that LCMV infection also blocks activation and transcriptional activity of NF-κB induced by SeV infection (). Since several virus-encoded IFN-I-antagonist proteins have been described to counteract two or more of the transcription factors involved in IFN-I production (19
), we examined whether LCMV-NP could also inhibit activation of NF-κB. Our results have shown that LCMV-NP, in the absence of any other viral protein, exerts a powerful dose-dependent inhibitory effect on NF-κB-mediated transcriptional activation upon SeV infection. In contrast, LCMV-NP had only a modest inhibitory effect on TNF-α-induced activation of NF-κB (). These results suggest that LCMV-NP is able to interfere with the nonclassical, but not the classical, pathway of NF-κB activation.
Most of the signaling pathways that lead to activation of NF-κB converge on the IKK kinase complex, which acts as a master regulator of NF-κB activation. TNF-α-induced activation of NF-κB is initiated by binding of TNF-α to its receptors, TNFR1 and TNFR2, which results in activation of IKKα and IKKβ of the IKK complex, as well as TBK-1 (11
), leading to phosphorylation and subsequent ubiquitination and proteosomal degradation of the IκBα subunit of the inhibitor IκB (31
). These events result in the release and nuclear translocation of NF-κB, its binding to NF-κB response elements, and expression of target genes. In contrast, SeV infection stimulates the cellular RNA sensors RIG-I and MDA5 that, via the mitochondrial antiviral signaling protein MAVS (15
) (also known as IPS-1/VISA/Cardif) (36
), activate IKKα and IKKβ of the IKK complex as well as the IKK-related kinases TBK-1 and IKKε, which have been associated with activation of IRF3/IRF7, as well as the NF-κB pathway (30
). Accordingly, we observed that SeV infection induced activation of the IFN-β- and NF-κB-dependent promoters, but the magnitude of the induction was higher with the IFN-β-dependent promoter (). In contrast, and consistent with the literature, TNF-α induced higher levels of activation of the NF-κB-dependent promoter and weaker activation of the IFN-β-dependent promoter (32
) (). In agreement with previous findings, we observed that transient transfection with either TBK-1 or IKKε promoted activation of both IFN-β (9
)- and NF-κB (3
)-dependent promoters ( and ). The NF-κB-dependent promoter within the reporter plasmid contains only two NF-κB binding sites, which explains the overall low levels of reporter expression following activation of the promoter. Notably, infection with vesicular stomatitis virus (VSV) was shown to result in recruitment of IKKε, but not TBK-1, by MAVS (45
). In the accompanying paper by Pythoud et al.
), we demonstrate that arenavirus NP strongly binds IKKε but not TBK-1. Moreover, preliminary data revealed a biochemical association of arenavirus NP with IKKα by the use of nonstringent coimmunoprecipitation conditions. Consequently, it is plausible to imagine a situation where arenavirus NPs block efficiently SeV-induced activation of NF-κB while exhibiting only a very limited ability to inhibit TNF-α-mediated activation of NF-κB. A more detailed understanding of arenavirus NP interactions with the various cellular kinases involved in NF-κB activation by viral infection and TNF-α would help to elucidate the mechanisms by which arenavirus NPs can interfere differently with these two key pathways of the host inflammatory response.
Fig 7 IFN-β and NF-κB promoter activation by TBK-1 and IKKε. (A and B) SeV infection (A) or TNF-α treatment (B) results in activation of both IFN-β- and NF-κB-dependent promoters. Plasmids pNF-κB-GFP (500 (more ...)
The ability to inhibit NF-κB activation was shared by many arenavirus NPs (). Interestingly, and as previously observed together with the inhibitory effect of arenavirus NPs on IRF3 activation (53
), TCRV-NP was significantly less efficient in inhibiting nuclear translocation of NF-κB and its transcriptional activity. Mutation-function studies using a series of C-terminal and internal deletion mutants, as well as single alanine (A) substitutions (), revealed that the C-terminal region of LCMV-NP, and, specifically, residues within the active site of the NP 3′-5′ exonuclease (D382, E384, D459A, H517A, and D522A), as well as the highly conserved residues G385 and R386 within the DIEGR motif previously shown to be involved in the anti-IFN-I activity of NP (23
), also affected the NP's ability to inhibit NF-κB transcriptional activity. Accordingly, both rLCMV/NP* D382A and TCRV exerted only a very modest inhibitory effect on SeV-induced NF-κB transcriptional activity (). Our findings determined using plasmid-based cell transfection assays were also recreated in LCMV-infected cells with endogenous NF-κB after SeV infection or TNF-α treatment ().
The pathogenicity of HF arenaviruses has been linked to an impaired host innate immune response and subsequent deficient adaptive immune responses, which result in uncontrolled high levels of viremia (55
). Thus, differences in pathogenicity between the two OW arenaviruses LASV (highly pathogenic) and Mopeia virus (MOPV [nonpathogenic]) have been correlated with LASV's ability to prevent the activation and corresponding cytokine production observed in MOPV-infected macrophages and dendritic cells (DCs) (47
). Whether or not MOPV-NP is able to counteract the IFN-I response remains to be determined, but recent evidence suggests that MOPV infection is not able to counteract the IFN-I response at levels comparable to those seen with LASV-infected cells (10
). Likewise, in PICV-infected guinea pigs, a surrogate animal model of LF, a lethal (P18) but not an attenuated (P2) variant of PICV caused inhibition of proinflammatory cytokine production early during infection (6
). This was found to correlate with the ability of the pathogenic PICV-P18 to induce increased amounts of the transcription-repressing p50/p50 homodimer of NF-κB, whereas infection with the attenuated PICV-P2 led to accumulation of the trans
-activating p65/p50 heterodimer of NF-κB (7
). PICV-P2 and -P18 have an arginine (R) and a lysine (K), respectively, at position 374 of NP, but the two NPs were equally able to inhibit SeV-mediated IFN-β promoter activation (43
), suggesting that this single amino acid substitution in PICV-NP is unlikely to be responsible for the observed differences between the attenuated P2 and the virulent P18 variants of PICV in NF-κB activation. Similarly, infection of human monocytes with the nonpathogenic NW arenavirus TCRV was accompanied by significant upregulation of interleukin-6 (IL-6), IL-10, and TNF-α compared to infection of monocytes with the pathogenic NW arenavirus JUNV (22
). These results are in agreement with our observation that TCRV-NP is not as efficient as other arenavirus NPs in blocking IFN-I (53
) and NF-κB responses (this work). Intriguingly, although increased production of IFN-α, IFN-β, TNF-α, IL-6, IL-10, or IL-12 was not observed in JUNV-infected human monocytes or macrophages, several pro- and anti-inflammatory cytokines have been shown to occur at elevated levels in patients with fatal or severe JUNV infection (25
), a situation similar to that observed in human cases of LASV infection (47
). However, studies of LASV infection of cynomolgus monkeys have shown a correlation between an early and strong innate immune response and control of virus multiplication and recovery from LASV-induced disease (34
). These findings support a model of arenavirus pathogenesis in which an early robust production of cytokine by infected monocytes and macrophages may be protective. This model is also consistent with gene expression data from a model of LF based on LCMV infection of macaques (12
). During previremic early stages of infection, LCMV had mainly an inhibitory effect on host gene expression, including expression of genes within the IFN-I and NF-κB pathways. However, upon the onset of viremia, the trend was reversed, and in macaques infected with the virulent WE strain of LCMV, there was an overall increase in host gene transcription (48
). One could envision that, early during infection, expression of NP might contribute to a limited induction of IFN-I and NF-κB activation in the infected cells. Over time, however, as the numbers of infected cells and the overall viral load rise, many noninfected cells could start to respond to the signals provided by infected cells, thus leading to an overall increase in host gene transcription.
NF-κB is also involved in the regulation of cellular apoptosis. In cultured cells, infection with TCRV, but not JUNV, has been shown to induce a robust cytopathic effect (22
). Interestingly, we observed that infection of A549 cells with rLCMV/NP* D382A, a virus whose NP lacked the ability to counteract induction of IFN-I, resulted in a high degree of cytopathic effect than was not seen in cells infected with wild-type rLCMV (data not shown). A similar situation has been observed with recombinant influenza viruses lacking the NS1 protein, which is known to counteract both induction of IFN-I and NF-κB activation (84
). It is plausible that the cytopathic effect observed with TCRV and rLCMV/NP* (D382A) could be related to their NPs being unable to modulate the activation of the NF-κB pathway. Further studies would be required to determine the role of arenavirus NP in cellular apoptosis.
Our results have uncovered a previously unknown function of arenavirus NP, namely, its ability to interfere with NF-κB activation, which could contribute to the multiple mechanisms by which arenaviruses counteract the host initial innate defenses and subsequent adaptive immune responses. A better knowledge of the mechanisms underlying the inhibitory activity of arenavirus NPs on the IFN-I and inflammatory responses would lead to a better understanding of the pathogenic and immunogenic properties of arenaviruses. Insights into arenavirus virulence may also open new avenues for the generation of highly attenuated arenavirus that could be evaluated as vaccine candidates and may suggest new antiviral targets for therapeutic modulation in the treatment of arenavirus infections.