Classical swine fever (CSF) caused by CSF virus (CSFV) leads to severe economic losses in pig industry especially in developing countries. The role of CSFV NS5A on the molecular level has been well characterized, but much less is known about the relevance of CSFV NS5A for CSFV-associated pathogenesis. To gain more insight in CSFV NS5A protein, this study was conducted to explore the effect of CSFV NS5A on inflammatory cytokines and its mechanisms. Eventually, the results showed that, CSFV NS5A could suppressed poly(I:C)-stimulated inflammatory cytokine secretion by suppressing the NF-?B signaling pathway.
Following recognition of viral RNA, RIG-I and MDA5 undergo conformational changes for signal propagation to activate downstream through interactions with IPS-1 adaptor protein, which serves to activate downstream IRF, NF-?B and other transcription factors [22]. In vitro studies suggest that both RIG-I and MDA5 detect poly(I:C), a synthetic dsRNA analogue [23]. NF-?B, a sequence specific transcription factor, can regulate the expression of numerous cellular and viral genes and plays important roles in cell survival, tumorigenesis, inflammation and innate immune responses. In resting cells, NF-?B stays inactive in the cytoplasm combined with its inhibitory subunit IkB?. After exposure to a variety of agonists, the activation of NF-?B occurs through the degradation of IkB? [24, 25]. CSFV NS5A protein has shown to be involved in viral replication [7–9]. A closely related functional viral protein to the CSFV NS5A is the HCV NS5A protein while HCV belongs to the same Flaviviridae family. HCV NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. Now, it is regarded as a new target for antiviral drugs in the treatment of HCV infection [26]. Recent reports have demonstrated that HCV NS5A protein exerts its functions through its regulation via cell signaling pathways such as STAT1 pathway [27], MEK/ERK pathway [28], a FoxO1-dependent pathway [29], and PKR-p38 pathway [30]. Furthermore, HCV NS5A over-expression significantly enhanced survivin transcription by increasing p53 degradation and stimulating NOS2A expression as well as NF-?B relocation to the nucleus [31]. HCV NS5A suppressed p53-mediated transcriptional transactivation and apoptosis during HCV infection [32], blocked poly(I:C) or interferon (IFN)-?-mediated IRF-7 nuclear translocation [33] or inhibited TNF-?-induced NF-?B activation in vitro [34]. Furthermore, HCV NS5A activated NF-?B through oxidative stress or tyrosine phosphorylation of IkB? and its degradation by calpain protease [35]. In the present study, we found that CSFV NS5A did not disrupt the expressions of RIG-I, MDA5, IPS-1 stimulated by poly(I:C) in PAMs. However, CSFV NS5A protein inhibited poly(I:C)-induced NF-?B nuclear translocation and activity, and IkB? degradation, which resulted in the suppression of inflammatory cytokine IL-1?, IL-6 and TNF-? secretion induced by poly(I:C).
Early detection of viruses by the innate immune system is critical for host defense. Antiviral immunity is first to be initiated by pattern recognition receptors (PRRs) that recognize viral pathogen-associated molecular patterns (PAMPs). Intracellular PRRs then stimulate the production of interferons and cytokines to orchestrate immune responses. The key host factors that are critical for antiviral immunity and for systemic inflammatory reactions include IL-1?, IL-6 and TNF-? [36]. TNF-a, IL-1 and IL-6 are three proinflammatory cytokines that form part of a complex defence network that protects the host against inflammatory agents, microbial invasion and injury [37]. IL secretion is necessary to stimulate immune cell responses and IL-1 is released from CSFV-infected macrophages [38]. Recent studies have demonstrated that the highly active proinflammatory cytokine IL-1? is essential in antiviral host defense. Despite its essential role in host defense, high levels of IL-1? are also responsible for unwanted effects like fever, vasodilatation, hypotension or acute lung injury by fluid accumulation in response to viral infection [39]. In the transgenic mouse model, HCV NS5A could impair both the innate and the adaptive immune response to promote chronic HCV infection [16] through the blockade of IFN-? induction by NS5B [40], the inhibition of interferon-alpha signaling [41], the competed binding to CypA [42], and a up-regulation of IL-8 [15]. The finding in vivo suggested that CSFV infection promoted serum levels of IFN-?, IL-8 and TNF-? in 6-month-old pigs, indicating the involvement of these cytokines in the immune response during CSFV infection with strains of different virulence [43]. Our previous study in vitro revealed that high virulent CSFV shimen strain could significantly promote the secretion of IFN-?, IFN-?, IL-1?, IL-6 and TNF-? through the activation of the RIG-I signaling pathway [44]. The present study further demonstrated that the stable expressed CSFV NS5A had no influence on the expressions of inflammatory cytokines IL-1?, IL-6 and TNF-? in PAMs without poly (I:C) stimulation. Moreover, CSFV NS5A protein could suppress IL-1?, IL-6 and TNF-? expression induced by poly (I:C).