Antiviral Activity of Canine RIG-I against Canine Influenza Virus and Interactions between Canine RIG-I and CIV

Abstract

RIG-I functions as a virus sensor that induces a cellular antiviral response. Although it has been investigated in other species, there have been no further studies to date on canine RIG-I against canine influenza virus (CIV). In the present study, we cloned the RIG-I gene of beagle dogs and characterized its expression, subcellular localization, antiviral response, and interactions with CIV proteins. RIG-I was highly expressed and mainly localized in the cytoplasm, with low levels detected in the nucleus. The results revealed that overexpression of the CARD domain of RIG-I and knockdown of RIG-I showed its ability to activate the RLR pathway and induced the expression of downstream interferon-stimulated genes. Moreover, overexpression of canine RIG-I suppressed the replication of CIV. The association between RIG-I and CIV was evaluated with the luciferase assay and by indirect immunofluorescence and bimolecular fluorescence complementation analyses. The results showed that CIV nonstructural protein 1 (NS1) can strongly suppress the RIG-I–mediated innate immune response, and the novel interactions between CIV matrix proteins (M1 and M2) and canine RIG-I were disclosed. These findings provide a basis for investigating the antiviral mechanism of canine RIG-I against CIV, which can lead to effective strategies for preventing CIV infection in dogs.

Transcriptome Analysis of Retinoic Acid-Inducible Gene I Overexpression Reveals the Potential Genes for Autophagy-Related Negative Regulation

“…However, we still know relatively little about cellular autophagy serving for RIG-I regulation. To investigate whether cellular autophagy was involved in the phenomenon in Figure 1, we detected the expression level of LC3B protein in MDCK cells protein samples with the same treatments as our previous study [18]. The overexpression of RIG-I against H3N2 CIV experiments showed that the ratio of LC3B-II/GAPDH was relatively higher when the Flag-RIG-I plasmid dose for transfection was more than 2500 ng (Figure 2A,B).…”

“…Our previous study identified the antiviral effect of canine RIG-I against canine influenza virus (CIV), whereby overexpression of canine RIG-I could strongly suppress the CIV replication in MDCK and HEK 293T cells [18]. However, we found that more than 2500 ng canine RIG-I overexpression reduced the antiviral effect against CIV.…”

“…In our previous study, canine RIG-I in MDCK cells played an essential role against canine influenza virus (CIV) infection [18]. Interestingly, an outcome of more than 2500 ng canine RIG-I overexpression showed a gradually decreasing antiviral effect against CIV, even though overexpressed canine RIG-I indeed suppressed the CIV replication in MDCK cells and HEK 293T cells [18]. Here, we performed the same experiments again, and the results indicated a similar trend to our previous study.…”

“…Our previous study indicated that canine RIG-I shares similar function domains with human RIG-I as a homologous gene. Canine RIG-I could trigger the antiviral effect in the human cell line [18]. Therefore, we chose the HEK 293T cells with high transfection efficiency for understanding the comprehensive regulations in cells transfected with exogenous RIG-I and revealing the potential genes for autophagy-related negative regulation by transcriptome analysis.…”

Reduction in Interferon-Stimulated Genes Contributes to High-Yield Production of Influenza Virus in Suspension MDCK Cells

“…For the influenza virus, Retinoic acid-inducible gene I (RIG-I, encoded by the DDX58 gene) is the major pathogen recognition receptor (PRR) of the cytoplasm to induce the production of type I interferon (IFN-α/β) and pro-inflammatory cytokines [ 19 ]. It has been reported that canine RIG-I suppresses the replication of the canine influenza virus (CIV) in adherent MDCK (aMDCK) cells [ 20 ]. The secreted IFN-α/β activates the JAK/STAT signaling pathway, leading to the induction of hundreds of IFN-stimulated genes (ISGs) including ISG15, MX1, PKR, OAS1, IFI44, and IFITM3 [ 21 , 22 , 23 , 24 , 25 ].…”