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Macrophages play a central role in the innate immune response to respiratory viral infections through pro-inflammatory factor secretion and phagocytosis. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize macrophage function. In our recent in vitro analyses of murine macrophage cell lines, Sendai virus (SeV) accessory protein C inhibited the secretion of pro-inflammatory factors, and C gene-knockout SeV (SeVΔC) caused drastic morphological changes in RAW264.7 macrophages, similar to those observed after stimulation with Lipid A, a well-known activator of actin-rich membrane ruffle formation and phagocytosis. Hence, we sought to determine whether the C protein limits phagocytosis in SeV-infected macrophages through the suppression of membrane ruffling. Phagocytosis assays indicated an upregulation of phagocytosis in both SeVΔC-infected and Lipid A-stimulated macrophages, but not in SeV WT-infected cells. Further, the observed membrane ruffling was associated with phagocytosis. RIG-I is essential for Lipid A-induced phagocytosis; its deficiency inhibited SeVΔC-stimulated phagocytosis and ruffling, confirming the essential role of RIG-I. Moreover, treatment with interferon (IFN)-β stimulation and neutralizing antibodies against IFN-β suggested that SeVΔC-induced phagocytosis and ruffling occurred in an IFN-β-independent manner. A newly isolated SeVΔC strain that does not generate dsRNA further highlighted the importance of dsRNA in the induction of phagocytosis and ruffling. Taken together, the current results suggest that SeV C protein might limit phagocytosis-associated membrane ruffling in an RIG-I-mediated but IFN-independent manner via limiting the generation of intracellular dsRNA.

Abstract Background Capsid (C) protein plays an important role in the replication of classical swine fever virus (CSFV). The ubiquitin proteasome system (UPS) involves in replication of many viruses via modulation of viral proteins. The relationship of CSFV with UPS is poorly understood and the impact of 26S proteasome on C protein has never been reported before. Methods In this study, fused C protein with an EGFP tag is expressed in PK-15 and 3D4/2 cells. MG132 and 3-methyladenine (3-MA) are used to detect the roles of 26S proteasome and autophagolysosome in expression levels of C protein. Truncated and mutant C proteins are used to find the exact residues responsible for the degradation of C protein. Immunoprecipitaion is performed to find whether C protein is ubiquitinated or not. Results C-EGFP protein expresses in a cleaved form at a low level and is degraded by 26S proteasome which could be partly inhibited by MG132. C-terminal residues play more important roles in the degradation of C protein than N-terminal residues. Residues 260 to 267, especially M260 and L261, are crucial for the degradation. In addition, C-terminal residues 262 to 267 determine cleavage efficiency of C protein. Conclusions CSFV C protein is degraded by 26S proteasome in a ubiquitin-independent manner. Last 8 residues at C-terminus of immature C protein play a major role in proteasomal degradation of CSFV C protein and determine the cleavage efficiency of C protein by signal peptide peptidase (SPP). Our findings provide valuable help for fully understanding degradation process of C protein and contribute to fully understanding the role of C protein in CSFV replication.