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. 2015 Oct;67(2 Pt B):607-15.
doi: 10.1016/j.molimm.2015.07.011. Epub 2015 Aug 5.

Duck TRIM27-L enhances MAVS signaling and is absent in chickens and turkeys

Alysson H Blaine  1 , Domingo Miranzo-Navarro  1 , Lee K Campbell  1 , Jerry R Aldridge Jr  2 , Robert G Webster  2 , Katharine E Magor  3
Affiliations

Duck TRIM27-L enhances MAVS signaling and is absent in chickens and turkeys

Alysson H Blaine et al. Mol Immunol. 2015 Oct.

Abstract

Wild waterfowl, including mallard ducks, are the natural reservoir of avian influenza A virus and they are resistant to strains that would cause fatal infection in chickens. Here we investigate potential involvement of TRIM proteins in the differential response of ducks and chickens to influenza. We examine a cluster of TRIM genes located on a single scaffold in the duck genome, which is a conserved synteny group with a TRIM cluster located in the extended MHC region in chickens and turkeys. We note a TRIM27-like gene is present in ducks, and absent in chickens and turkeys. Orthologous genes are predicted in many birds and reptiles, suggesting the gene has been lost in chickens and turkeys. Using quantitative real-time PCR (qPCR) we show that TRIM27-L, and the related TRIM27.1, are upregulated 5- and 9-fold at 1 day post-infection with highly pathogenic A/Vietnam/1203/2004. To assess whether TRIM27.1 or TRIM27-L are involved in modulation of antiviral gene expression, we overexpressed them in DF1 chicken cells, and neither show any direct effect on innate immune gene expression. However, when co-transfected with duck RIG-I-N (d2CARD) to constitutively activate the MAVS pathway, TRIM27.1 weakly decreases, while TRIM27-L strongly activates innate immune signaling leading to increased transcription of antiviral genes MX1 and IFN-β. Furthermore, when both are co-expressed, the activation of the MAVS signaling pathway by TRIM27-L over-rides the inhibition by TRIM27.1. Thus, ducks have an activating TRIM27-L to augment MAVS signaling following RIG-I detection, while chickens lack both TRIM27-L and RIG-I itself.

Keywords: Avian immunology; Evolution; Influenza A; Innate immune modulation; MAVS signaling pathway; TRIM27; Tripartite-motif family.

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Figures

Fig. 1
Fig. 1
Organization of the TRIM gene cluster adjacent to avian MHC regions. The gene organization implies the locus is a conserved synteny group of duck TRIM genes compared to the chicken and turkey MHC adjacent TRIM clusters (Ruby et al. 2005; Chaves et al. 2009). The duck genes are on Ensembl scaffold #KB742989.1.
Fig. 2
Fig. 2
Phylogenetic tree of TRIM27 related genes. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [1]. The tree with the highest log likelihood (−17555.5723) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using a JTT model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 2.0142)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 1.4941% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 57 amino acid sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 394 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.02 (release # 6140226) (Tamura et al. 2011).
Fig. 3
Fig. 3
Transcript abundance for duck TRIM27.1 and TRIM27-L is increased in lung tissue following infection with highly pathogenic VN1203. Total RNA was extracted from duck tissues harvested at 1, 2 and 3 dpi with BC500 and VN1203, cDNA was synthesized and analyzed with qPCR using gene specific probe and primers for targets and endogenous control (GAPDH). Fold expression of TRIM27.1 (A) and TRIM27-L (B) in duck lung tissues is shown relative to a mock-infected animal on each day. Dots represent individual ducks (n = 3) and the values are the mean fold change relative to expression level for a mock animal each day (*** is p <0.001).
Fig. 4
Fig. 4
TRIM27.1 and TRIM27-L expression have opposite effects on transcription of antiviral genes downstream of MAVS signalling. Duck TRIM27.1 reduced expression of (A) IFN-β and (B) MX1 in chicken DF-1 cells activated with the RIG-I N-terminal domain (d2CARD), which constitutively activates MAVS signalling. TRIM27-L increased (C) IFN-β and (D) MX1 expression in cells activated with d2CARD. Each unique symbol represents data from four independent transfection experiments, made with triplicate samples. GST-I is an irrelevant protein control for d2CARD-GST.
Fig. 5
Fig. 5
TRIM27.1 suppresses and TRIM27-L enhances MAVS signalling. Four replicate dual-luciferase reporter co-transfection experiments with the IFN-β promoter, d2CARD and increasing concentrations of plasmids corresponding to (A) TRIM27.1 or (B) TRIM27-L or (C) increasing concentration of TRIM27-L co-transfected with TRIM27.1 were done in DF-1 cells. A dose-dependent increase in IFN-β promoter activity is observed with increasing amounts of TRIM27-L, even in presence of TRIM27.1. Different symbols indicate results from independent transfection experiments.
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