Arenaviruses cause chronic infections of rodent species with a worldwide distribution (8). Human infections, which occur via mucosal exposure to aerosols or by direct contact with infectious materials, can cause severe disease, including hemorrhagic fever (HF). Thus, Lassa virus (LASV) and Junin virus (JUNV) are the causative agents of Lassa fever (LF) and Argentine HF disease, respectively, which represent significant public health problems within their geographic regions of endemicity of West Africa (LASV) and Argentina (JUNV) (8, 55, 68, 95). In addition, evidence indicates that the globally distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance (2, 35, 57) and poses a special threat to immunocompromised individuals (16, 64). Public health concerns posed by arenaviruses are aggravated by the lack of Food and Drug Administration (FDA)-licensed vaccines and current antiarenavirus therapy being limited to an off-label use of the nucleoside analog ribavirin that is only partially effective and associated with significant side effects (37, 56, 60, 76, 83, 94). Therefore, it is important to develop novel and effective antiviral strategies to combat human-pathogenic arenaviruses. Morbidity and mortality associated with LASV infection, and other HF arenavirus infections, have been associated with the failure of the host's innate immune response to restrict virus replication and to facilitate the initiation of an effective adaptive immune response (55). Likewise, chronic infection of the prototypic arenavirus LCMV in mice, the natural reservoir of LCMV, has been shown to be associated with only a very modest increase in production of type I interferon (IFN-I), despite levels of viral RNA being readily detected in most tissues and cell types of the infected host. These findings suggest a virus's ability to counteract the activity of host pattern recognition receptors (PRRs) that sense the presence of viral RNA (78). Accordingly, the arenavirus NP has been shown to inhibit the host's IFN-I response via an early blockade in the IFN-I regulatory factor 3 (IRF3) activation pathway, thereby inhibiting IFN-I production and subsequent induction of IFN-I-stimulated genes (ISGs) (54). The anti-IFN-I activity of LCMV-NP was mapped to its C-terminal region (residues 370 to 553), including the DIEGR motif (amino acid residues 382 to 386) (52). The determination of the X-ray crystal structure of LASV-NP (23, 73) identified a 3′-5′ exonuclease domain within the C-terminal region of the protein, with amino acid residues corresponding to positions D382, E384, D459, H517, and D522 in LCMV-NP contributing to its active site. Mutation-function studies established a link between the exonuclease and anti-IFN-I activities of LASV-NP (23, 52, 73). The exact mechanisms by which arenavirus NPs modulate the IFN-I response still remain unclear, but retinoic acid-inducible gene I (RIG-I)/melanoma-differentiation-associated gene 5 (MDA5) pathway-mediated IFN-I production appears to be involved (97).

Induction of production of the different IFN-I forms upon viral infection involves multiple transcription factors. Early activation of the beta IFN (IFN-β) promoter involves the cooperation of IRF3 with NF-κB and activator protein 1 (AP-1), whereas later induction of IFN-α forms is mainly dependent on IRF7 and, to a lesser extent, IRF3 (20, 28, 31). NF-κB comprises a family of transcription factors found ubiquitously in numerous cell types that play important roles in host inflammatory and immune responses, cell growth and cell differentiation, and apoptosis (28, 31). Under normal conditions, NF-κB is bound to its inhibitor, IκB, resulting in its cytoplasmic retention. Activation of NF-κB signaling requires phosphorylation of IκB by the IκB kinase (IKK) complex (31, 33). The IKK complex contains two catalytic kinase components, IKKα and IKKβ, as well as a nonenzymatic regulatory subunit, NEMO. Two additional members of the family of IKK kinases (TANK-binding kinase 1 [TBK-1] and IKKε) also have the potential to stimulate NF-κB signaling (28, 31). Upon activation, IκB kinase complexes phosphorylate IκB, which is subsequently ubiquitinated and undergoes proteosomal degradation (1). NF-κB is thereby released and enters the nucleus, where it stimulates transcription of genes containing NF-κB-binding sequence elements in their promoters. NF-κB can be activated by various stimuli, including tumor necrosis factor alpha (TNF-α) (28, 31), which is a major proinflammatory cytokine, produced by a variety of cell types, including macrophages, endothelial cells, and epithelial cells (89), and with multiple roles in the regulation of immune response, inflammation, cellular differentiation, apoptosis, and host antiviral defense (27). TNF-α engagement to its receptor leads to signaling cascades that activate IKK complexes (24, 27, 90). NF-κB can also be activated in response to viral infections (14, 91). Because of the importance of NF-κB in the induction of IFN-I and proinflammatory molecules, many viruses have developed mechanisms to evade NF-κB activation (21, 31, 85). Virus-encoded proteins that block NF-κB activation, including influenza virus nonstructural protein 1 (NS1) (91), Hantaan virus (HTNV) nucleocapsid protein (N) (86), and measles virus (MV) V protein (80), have been shown to enhance viral replication in vitro and in vivo and contribute to virus pathogenesis (28, 31).

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 (Fig. 1). 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, 31, 92, 93), 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 (Fig. 2). 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, 42), leading to phosphorylation and subsequent ubiquitination and proteosomal degradation of the IκBα subunit of the inhibitor IκB (31, 33). 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, 58, 71, 96), 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, 41). 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 (Fig. 7A). 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, 46, 74) (Fig. 7B). In agreement with previous findings, we observed that transient transfection with either TBK-1 or IKKε promoted activation of both IFN-β (9, 17, 26, 38, 67, 81)- and NF-κB (3, 18, 40, 62, 69, 70, 77, 79, 82, 87)-dependent promoters (Fig. 7C and ​andD).D). 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. (72), 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.

The ability to inhibit NF-κB activation was shared by many arenavirus NPs (Fig. 3). 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 (Fig. 4), 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, 52, 73), 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 (Fig. 5). 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 (Fig. 6).

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, 65). 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, 44, 50), 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, 49). 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.