Classical swine fever virus (CSFV) non-structural protein 3 (NS3) is a multifunctional non-structural protein that plays a major role in viral replication. However, how exactly NS3 exerts these functions remains unknown. Here, we identified tumour necrosis factor receptor-associated factor 6 (TRAF6) as a novel NS3-interacting protein via yeast two-hybrid analysis, co-immunoprecipitation, and glutathione S-transferase pull-down assays. Furthermore, we observed that TRAF6 overexpression significantly inhibited CSFV replication, and TRAF6 knockdown promoted CSFV replication in porcine alveolar macrophages. Additionally, TRAF6 was degraded during CSFV infection or NS3 expression exclusively, indicating that CSFV and TRAF6 were mutually antagonistic and that TRAF6 degradation might contribute to persistent CSFV replication. Moreover, nuclear factor-kappa B (NF-κB) activity and interferon (IFN)-β and interleukin (IL)-6 expression were increased in TRAF6-overexpressing cells, whereas TRAF6-knockdown cells exhibited decreased NF-κB activity and IFN-β and IL-6 levels. Notably, TRAF6 overexpression did not reduce CSFV replication following inhibition of NF-κB activation by p65 knockdown. Our findings revealed that TRAF6 inhibits CSFV replication via activation of NF-κB-signalling pathways along with increases in the expression of its targets IFN-β and IL-6. This work addresses a novel aspect concerning the regulation of innate antiviral immune response during CSFV infection.
Virus infection may induce excessive interferon (IFN) responses that can lead to host tissue injury or even death. β-arrestin 2 regulates multiple cellular events through the G protein-coupled receptor (GPCR) signaling pathways. Here we demonstrate that β-arrestin 2 also promotes virus-induced production of IFN-β and clearance of viruses in macrophages. β-arrestin 2 interacts with cyclic GMP-AMP synthase (cGAS) and increases the binding of dsDNA to cGAS to enhance cyclic GMP-AMP (cGAMP) production and the downstream stimulator of interferon genes (STING) and innate immune responses. Mechanistically, deacetylation of β-arrestin 2 at Lys171 facilitates the activation of the cGAS–STING signaling and the production of IFN-β. In vitro, viral infection induces the degradation of β-arrestin 2 to facilitate immune evasion, while a β-blocker, carvedilol, rescues β-arrestin 2 expression to maintain the antiviral immune response. Our results thus identify a viral immune-evasion pathway via the degradation of β-arrestin 2, and also hint that carvedilol, approved for treating heart failure, can potentially be repurposed as an antiviral drug candidate.
Classical swine fever (CSF) is a severe, febrile and highly contagious disease caused by classical swine fever virus (CSFV) that has resulted in huge economic losses in the pig industry worldwide. CSFV Npro has been actively studied but remains incompletely understood. Few studies have investigated the cellular proteins that interact with Npro and their participation in viral replication. Here, the yeast two-hybrid (Y2H) system was employed to screen Npro-interacting proteins from a porcine alveolar macrophage (PAM) cDNA library, and a blast search of the NCBI database revealed that 15 cellular proteins interact with Npro. The interaction of Npro with ribosomal protein S20, also known as universal S10 (uS10), was further confirmed by co-immunoprecipitation and glutathione S-transferase pull-down assays. Furthermore, uS10 overexpression inhibited CSFV replication, whereas the knockdown of uS10 promoted CSFV replication in PAMs. In addition, Npro or CSFV reduced uS10 expression in PAMs in a proteasome-dependent manner, indicating that Npro-uS10 interaction might contribute to persistent CSFV replication. Our previous research showed that CSFV decreases Toll-like receptor 3 (TLR3) expression. The results showed that uS10 knockdown reduced TLR3 expression, and that uS10 overexpression increased TLR3 expression. Notably, uS10 knockdown did not promote CSFV replication following TLR3 overexpression. Conversely, uS10 overexpression did not inhibit CSFV replication following TLR3 knockdown. These results revealed that uS10 inhibits CSFV replication by modulating TLR3 expression. This work addresses a novel aspect of the regulation of the innate antiviral immune response during CSFV infection.
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