Grass carp Trim47 restricts GCRV infection via SPRY domain-mediated autophagic degradation of nonstructural proteins and disruption of viral inclusion bodies

Abstract

Trim47, a TRIM C-VII subgroup protein characterized by a conserved SPRY domain, has been primarily studied for its ubiquitin-dependent roles in mammals. This study reports a paradigm-shifting finding in teleost immunology: grass carp Trim47 (gcTrim47) employs its SPRY domain to execute a novel, ubiquitin-independent antiviral pathway, selectively degrading GCRV-I nonstructural proteins NS38/NS80 via autophagy-mediated clearance. Unlike mammalian TRIMs, gcTrim47 antiviral activity is strictly dependent on its SPRY domain-devoid of RING/B-box domains critical for E3 ligase function-revealing an evolutionarily divergent mechanism where substrate-targeting specificity, not ubiquitination, drives viral replication factory (viral inclusion body, VIB) dismantling. Functional assays demonstrated that gcTrim47 overexpression in CIK cells reduced viral titers and suppressed VIB formation, with SPRY domain deletion ablating these effects. In vivo, a yeast surface-display platform presenting gcTrim47-PYD1 conferred 32.94% relative percent survival (RPS) against GCRV-II infection, the first reported use of a TRIM family protein as an antiviral immunogen in grass carp. This strategy mitigated splenic/kidney viral loads and alleviated histopathological damage, including tubular necrosis and inflammatory infiltration. The successful application of this mechanism into a yeast-based immunization strategy highlights its potential for developing novel antiviral biotherapeutics in aquaculture.

Keywords: SPRY domain; autophagic degradation; gcTRIM47-PYD1 recombinant Saccharomyces cerevisiae biologics; grass carp Trim47; grass carp reovirus.

Figure 1

Effect of gcTrim47 on GCRV replication. (A, B) Crystal violet staining (A) and plaque assay-based quantification of GCRV titers (B) were performed in CIK cells transfected with FLAG or gcTrim47-FLAG and infected with GCRV at a MOI of 1. (C) Expression levels of GCRV structural proteins (VP3, VP5) and nonstructural proteins (NS38, NS80) were analyzed in CIK cells transfected with FLAG or gcTrim47-FLAG and infected with GCRV at a MOI of 1. Protein band intensities were quantified using Image J software for densitometric analysis. ***p < 0.001.

Figure 2

gcTrim47 degrades GCRV non-structural proteins in a SPRY domain-dependent manner. (A) Schematic diagrams of different domain-deletion constructs of gcTrim47. (B) The effect of different domain-deletion constructs of gcTrim47 on the endogenous expression of GCRV non-structural proteins NS80 and NS38. (C, D) The effect of different domain-deletion constructs of gcTrim47 on the exogenous expression of GCRV non-structural proteins NS80 or NS38. (E) Schematic diagrams of gcTrim47 and gcTrim47-SPRY (a mutant containing only the SPRY domain). (F) The effect of gcTrim47-SPRY on the endogenous expression of GCRV non-structural proteins NS80 and NS38. Protein band intensities were quantified using Image J software for densitometric analysis.

Figure 3

gcTrim47 promotes degradation of GCRV nonstructural proteins through the autophagy pathway. (A) Evaluation of gcTrim47-mediated degradation of GCRV nonstructural proteins NS80 and NS38 in the presence of ubiquitin-proteasome (MG-132) and autophagy-lysosome (3-MA and NH₄Cl) pathway inhibitors. (B) Interaction analyses between full-length gcTrim47 or its domain-deleted constructs and GCRV nonstructural proteins in CIK cells infected with GCRV-I. (C, D) Confocal microscopy analysis of subcellular co-localization between gcTrim47/domain-deleted constructs and GCRV nonstructural proteins NS80 (C) or NS38 (D) in GCRV-infected CIK cells. (E) The effect of different domain-deletion constructs of gcTrim47 on the endogenous expression of p62. (F) The subcellular colocalization between gcTrim47/domain-deleted constructs and autophagosomes (labeled by gcLC3-GFP). (G) The effect of different domain-deletion constructs of gcTrim47 on the autophagosome formation. Protein band intensities were quantified using Image J software for densitometric analysis.

Figure 4

gcTrim47 inhibits the formation of VIBs and GCRV infection in a SPRY domain-dependent manner. (A, B) Immunofluorescence analysis of VIB formation using anti-NS80 (A) or anti-NS38 (B) antibodies, evaluating the impact of full-length gcTrim47 and its domain-specific mutants. (C, D) Quantitative analysis of average VIB fluorescence intensity, assessed via anti-NS80 (C) or anti-NS38 (D) antibody staining. (E, F) The effect of different domain-deletion constructs of gcTrim47 on the GCRV infection, assessed via crystal violet staining (E) and plaque assay-based quantification of GCRV titers (F). ***p < 0.001.

Figure 5

Construction of the gcTrim47-PYD1/EBY100 yeast surface display system and in vivo GCRV-II infection model. (A) Western blot analysis of cell lysates from S. cerevisiae EBY100 expressing either PYD1 or gcTrim47-PYD1, confirming fusion protein expression. (B) Schematic depiction of the intraperitoneal immunization protocol followed by GCRV-II challenge in grass carp. (C) Comparative analysis of clinical manifestations in grass carp following GCRV-II infection.

Figure 6

Protective efficacy of engineered S. cerevisiae expressing gcTrim47 against GCRV-II infection in grass carp. (A) Assessment of the survival rate of grass carp following GCRV-II infection, comparing groups treated with engineered S. cerevisiae expressing gcTrim47 and control groups. (B, C) Quantification of the GCRV-II S11 gene copy numbers in the liver, spleen, and kidney tissues of grass carp. These tissues were collected from fish immunized with PYD1 or gcTrim47-PYD1 and infected with GCRV-II. (D) The constitutive expression of gcTrim47 in different tissues, including brain, heart, intestine, muscle, spleen, liver, gill, skin, blood and kidney. (E) The inducible expression of gcTrim47 in the liver, spleen, and kidney from grass carp infected with GCRV-II. These 3 tested tissues were collected at 6, 12 and 24 hpi. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Histopathological evaluation of spleen and kidney tissues in GCRV-II-infected grass carp. (A) Renal histology of GCRV-II-infected grass carp; (B) Splenic histology of GCRV-II-infected grass carp. (a, b) PYD1 control group; (c, d) gcTrim47-PYD1 immune group.