Objective The polyubiquitin chain linked to lysine (Lys) residues at 48^th position of ubiquitination modification chain of retinoic acid inducible gene I (RIG-I) regulates RIG-I protein stability to prevent over-activation of RIG-I signaling and host antiviral responses. The aim of the study was to explore whether bluetongue virus (BTV) also regulated RIG-I signaling by affecting ubiquitination modification of RIG-I for its own reproductive benefit.

Method The immortalized sheep pulmonary artery endothelium (SPAE) cells were infected with BTV, and then were treated with the proteasome inhibitor MG-132 and the deubiquitinase (DUB) inhibitor PR-619, respectively. The transcriptional levels of ring finger protein 125 (RNF125), ubiquitin-specific protease 4 (USP4), RIG-I, interferon regulatory factor 3 (IRF3), and interferon α (IFN-α), along with the genomic copy numbers of BTV were detected using RT-qPCR. The expression levels of proteins mentioned above were detected with Western blotting and ELISA. Immunofluorescence were conducted to analyze the nuclear translocation ratio of IRF3.

Result BTV infection upregulated the transcriptional levels of RNF125, RIG-I, IRF3, and IFN-α from 1.20 to 8.68-fold, and expression levels from 0.06 to 3.94-fold, respectively. The transcriptional level of USP4 gene slightly increased, but the expression level of USP4 was downregulated. Treatment with the proteasome inhibitor MG-132 significantly suppressed RIG-I degradation induced by BTV infection; The nuclear translocation ratio of IRF3 in MG-132 treated SPAE cells increased by 9.67 and 8.66 percentage points compared with their untreated counterparts at 24 hours post-infection (24 hpi) and 48 hpi; The expression level of IFN-α increased by 2.18-fold comparing with that of the corresponding untreated group at 48 hpi; The genomic copy numbers of BTV decreased to 74% and 85% of those of the untreated counterparts at 24 and 48 hpi, respectively. DUB inhibitor PR-619 obviously promoted RIG-I degradation; The nuclear translocation ratio of IRF3 in PR-619 treated SPAE cells decreased by 8.00 and 16.67 percentage points compared with their untreated counterparts at 24 and 48 hpi, respectively; The expression level of IFN-α decreased to 56.50% comparing with that of the corresponding untreated group at 24 hpi; The copy numbers of BTV genome increased to 1.93- and 1.49-fold of the untreated counterparts at 24 and 48 hpi, respectively.

Conclusion BTV utilized the ubiquitin-proteasome system (UPS) to regulate host RIG-I signaling to favor viral propagation.