Teleost-Specific TLR19 Localizes to Endosome, Recognizes dsRNA, Recruits TRIF, Triggers both IFN and NF-κB Pathways, and Protects Cells from Grass Carp Reovirus Infection

Abstract

TLRs are pivotal pattern recognition receptors in initiating innate immunity and triggering adaptive immunity. TLR pathways have been comprehensively investigated in mammals. However, the teleost-specific TLR19 pathway remains largely unknown. In this study, we identified TLR19 from grass carp (), and explored the ligand, adaptor, and signaling pathways. Pathogen-associated molecular pattern binding and luciferase activity assays indicate that TLR19 recognizes and responds to dsRNA analog (polyinosinic:polycytidylic acid). Confocal fluorescence microscopy demonstrates that TLR19 is synthesized in ribosomes not binding on endoplasmic reticulum, then transfers to early endosome post-polyinosinic:polycytidylic acid stimulation. Fluorescence colocalization and immunoprecipitation experiments confirm TLR19 interacts with adaptor TRIF, not MyD88, TIRAP, or SARM1. TLR19 facilitates protein and phosphorylation levels of IRF3, inhibits phosphorylation of IRF7. TLR19 enhances the promoter activities and mRNA expressions of major IFNs and NF-κBs; in contrast, grass carp TLR3 just significantly motivates IFN1 expression post-grass carp reovirus (GCRV) infection. Further investigations reveal that TLR19 inhibits GCRV replication by overexpression, knockdown, Western blotting techniques and virus titer assays, and protects cells from GCRV infection by flow cytometry and MTT method. Collectively, these results demonstrate that teleost-specific TLR19 recognizes dsRNA, recruits adaptor molecule TRIF, enhances IRF3 protein and phosphorylation levels, triggers both IFN and NF-κB pathways, and prevents viral proliferation. This is the first attempt to systematically clarify the TLR19 signaling pathway, which is the third TLR member recognizing dsRNA. The results will serve the antiviral immune mechanisms in teleost and evolutionary immunology.

Zebrafish prmt7 negatively regulates antiviral responses by suppressing the retinoic acid‐inducible gene‐I‐like receptor signaling

“…The interferon-stimulated response element (ISRE) luciferase reporter, two type 1 IFN promoter luciferase reporters (Dr-IFNφ1-Luc. 35 These findings suggest that IFN induction by poly (I: C) treatment or SVCV and GCRV infection is inhibited by prmt7 overexpression. and the EPC IFN promoter luciferase reporter are commonly used to monitor viral infection or poly(I:C) stimulation.…”

“…by GCRV infection in CIK cells ( Figure 2I,J). 35 These findings suggest that IFN induction by poly (I: C) treatment or SVCV and GCRV infection is inhibited by prmt7 overexpression.…”

“…Similarly, the overexpression of prmt7 also resulted in the reduction of ifn, isg15, and viperin expression induced by SVCV or GCRV infection in F I G U R E 1 Zebrafish prmt7 is inhibited after challenging with SVCV or GCRV. In addition, in CIK cells, we examined the expression of ifn1 (Ci-IFN1), ifi56 (Ci-IFN56), and mx2 (CiMX2), which are three typical IFN-stimulated genes in CIK cells, 35 upon poly(I:C) stimulation and GCRV (genotype I) infection. We injected 2-month-old zebrafish with SVCV (~2.51 × 10 7 TCID 50 /mL) or GCRV (genotype II) (~3.16 × 10 7 TCID 50 /mL) using 10 μL/individual.…”

Interleukin-17 suppresses grass carp reovirus infection in Ctenopharyngodon idellus kidney cells by activating NF-κB signaling

“…However, because of its large with a long reproductive cycle, it is hard to study the antiviral mechanisms in the grass carp in vivo (Lin et al, 2019). CIK cells can be infected by GCRV, and this infection is characterized by the systematic induction of a mild host immune response, involving a variety of classical antiviral signal transduction pathways (Ji et al, 2018;Yu et al, 2017;He et al, 2017). In our previous study, CIK cells were used to study the RNA expression patterns in response to viral infection, including circRNAs, mRNAs, and miRNAs.…”

Cyprinid viral diseases and vaccine development

“…TLR signaling pathways also participate in the immune responses to GCRV infection. Up-regulations of TLR3, TLR7, TLR8, TLR19, TLR22 and other TLRs are observed post poly(I:C) or GCRV challenge [115][116][117][118][119]. The enhancement of NOD1 and NOD2 also occur in grass carp post GCRV infection [120,121].…”