Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication

Abstract

Tumor necrosis factor receptor-associated factors (TRAFs) play important roles in the biological processes of immune regulation, the inflammatory response, and apoptosis. TRAF4 belongs to the TRAF family and plays a major role in many biological processes. Compared with other TRAF proteins, the functions of TRAF4 in teleosts have been largely unknown. In the present study, the TRAF4 homologue (EcTRAF4) of the orange-spotted grouper was characterized. EcTRAF4 consisted of 1413 bp encoding a 471-amino-acid protein, and the predicted molecular mass was 54.27 kDa. EcTRAF4 shares 99.79% of its identity with TRAF4 of the giant grouper (E. lanceolatus). EcTRAF4 transcripts were ubiquitously and differentially expressed in all the examined tissues. EcTRAF4 expression in GS cells was significantly upregulated after stimulation with red-spotted grouper nervous necrosis virus (RGNNV). EcTRAF4 protein was distributed in the cytoplasm of GS cells. Overexpressed EcTRAF4 promoted RGNNV replication during viral infection in vitro. Yeast two-hybrid and coimmunoprecipitation assays showed that EcTRAF4 interacted with the coat protein (CP) of RGNNV. EcTRAF4 inhibited the activation of IFN3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). Overexpressed EcTRAF4 also reduced the expression of interferon (IFN)-related molecules and pro-inflammatory factors. Together, these results demonstrate that EcTRAF4 plays crucial roles in RGNNV infection.

Keywords: Epinephelus coioides; RGNNV; TRAF4; cellular localization; viral replication.

Figure 1

Putative conserved domains of EcTRAF4. One RING domain, three zinc finger domains, and one MATH domain were predicted by SMART (http://smart.embl-heidelberg.de/, accessed on 1 May 2021).

Figure 2

Phylogenetic analysis of the EcTRAF4 proteins. The GenBank accession number for each species is listed to the left of the species name. The phylogenetic tree was constructed using MEGA software 4.0. The relationships among the various components were analyzed by the neighbor-joining (NJ) method. Numbers on the branches indicate percent bootstrap confidence values from 1000 replicates.

Figure 3

Tissue distribution of EcTRAF4 in healthy grouper. β-actin was used as the internal control. The expression of EcTRAF4 in the head kidney was set to 1.0. Data are expressed as the mean fold change (means ± S.E., n = 3) from the head kidney.

Figure 4

Subcellular localizations of EcTRAF4 in GS cells. GS cells were transfected with the plasmids of pEGFP–C1 and pEGFP–EcTRAF4 separately and then stained with DAPI. Samples were observed under fluorescence microscopy.

Figure 5

Effect of EcTRAF4 overexpression on RGNNV replication. (A) EcTRAF4 overexpression increased RGNNV gene transcription. Expression levels of CP and RdRp were measured using qRT-PCR (means ± S.E., n = 3). Statistical difference is with each pcDNA3.1-3HA-transfected cell. Asterisk denotes a significant difference (* p < 0.05 and ** p < 0.01). (B) Virus protein level after transfection with EcTRAF4. The level of RGNNV-CP was detected by western blot, and β-Tubulin was used as the internal control.

Figure 6

Effect of EcTRAF4 knockdown on RGNNV replication. (A) Three siRNA sequences were designed based on the sequence of EcTRAF4, the expression of EcTRAF4 was tested, and EcTRAF6 was used as the control (means ± S.E., n = 3). (B) Decreased RGNNV gene transcription, including CP and RdRp (means ± S.E., n = 3). Statistical difference is with each NC at a different time. Asterisk denotes a significant difference (* p < 0.05 and ** p < 0.01) (C) Virus protein level after transfection with siRNA1 of EcTRAF4. The level of RGNNV-CP was detected by western blot, and β-Tubulin was used as the internal control.

Figure 7

Interaction of CP with EcTRAF4. (A) For yeast two-hybrid analysis, pGBKT7-CP and pGADT7-EcTRAF4 plasmids were constructed for interaction verification. The two plasmids were co-transformed into S. cerevisiae strain Y2H Gold. The transformants were tested on a non-selective medium plate SD/-leu/-trp (DDO/X) to check whether the transformation was successful and the selective medium plate SD/-leu/-trp/-his/-ade/X-α-gal/AbA (QXA) to detect whether there was interaction between two proteins. (B) Co-immunoprecipitation resulted in GS cells showing that EcTRAF4 might interact with CP.

Figure 8

RGNNV and CP promoted EcTRAF4 expression. (A) Expression changes of EcTRAF4 in RGNNV-infected cells (means ± S.E., n = 3). β-actin was used as the internal control. Statistical difference is with mock. (B) EcTRAF4 mRNA in CP-expressing GS. GS were transfected with CP. TRAF4 mRNA expression was analyzed by RT–qPCR at 12 and 24 h. Asterisk denotes a significant difference (* p < 0.05 and ** p < 0.01).

Figure 9

The relative luciferase activity of IFN, ISRE, and NF-κB promoter in EcTRAF4-overexpressing cells. GS cells were co-transfected with NF-κB-Luc/IFN-β-Luc/ISRE-Luc, pEGFP-C1, and pEGFP-EcTRAF4. Twenty-four hours after transfection, cells were infected with RGNNV or left uninfected for 20 h before luciferase assays were performed (means ± S.E., n = 3). Asterisk denotes a significant difference (* p < 0.05 and ** p < 0.01).

Figure 10

Relative expression levels of IFN-signaling molecules and proinflammatory cytokines in EcTRAF4-overexpressing cells. (A) Expression changes of IFN-signaling molecules (ISG15, ISG56 and IFN-2) in EcTRAF4-overexpressing cells (means ± S.E., n = 3). (B) Expression changes of inflammatory factors (IL-1β and TNFα) in EcTRAF4-overexpressing cells (means ± S.E., n = 3). β-actin was used as the internal control. The asterisk denotes a significant difference (* p < 0.05 and ** p < 0.01).