Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C)

Abstract

Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) are viral RNA sensors that regulate host interferon (IFN)-mediated antiviral signaling. LGP2 (laboratory genetics and physiology 2) lacks the N-terminal caspase activation and recruitment domains (CARDs) responsible for signaling transduction in the other two RLR proteins, RIG-I and melanoma differentiation associated gene-5 (MDA5). How LGP2 regulates IFN signaling is controversial, and inconsistent results have often been obtained in overexpression assays when performed in fish cells and mammalian cells. Here we report that the differential sensitivity of fish cells and mammalian cells to poly(I:C) transfection conceals the function conservation of zebrafish and human LGP2. In fish cells, overexpression of zebrafish or human LGP2 initially activates IFN signaling in a dose-dependent manner, followed by inhibition at a critical threshold of LGP2 expression. A similar trend exists for LGP2-dependent IFN induction in response to stimulation by low and high concentrations of poly(I:C). In contrast, overexpression of zebrafish or human LGP2 alone in mammalian cells does not activate IFN signaling, but co-stimulation with very low or very high concentrations of poly(I:C) shows LGP2-dependent enhancement or inhibition of IFN signaling, respectively. Titration assays show that LGP2 promotes MDA5 signaling in mammalian cells mainly under low concentration of poly(I:C) and inhibits RIG-I/MDA5 signaling mainly under high concentration of poly(I:C). Our results suggest that fish and human LGP2s switch regulatory roles from a positive one to a negative one in increasing concentrations of poly(I:C)-triggered IFN response.

Coprophagia in early life tunes expression of immune genes after weaning in rabbit ileum

“…3 ). Signaling pathways downstream of these PRR activate “interferon regulatory factors” (IRF), and DExH-box helicase 58 (LGP2/DHX58), which acts as a regulator of DDX58/RIG-I and IFIH1 18 . IRF and DHX58 thus regulates the production of type I IFN 17 .…”

A protocol to characterize zebrafish LGP2 as a dual regulator of IFN response during viral infection

“…Overexpression of zebrafish LGP2 in fish cells provokes a strong IFN response at low doses but a weak one at high doses, indicating that the function switch of zebrafish LGP2 can be determined by luciferase assays through titration of zebrafish LGP2 itself ( Figure 4 ). Similar dual regulation of human LGP2 can be detected in mammalian cells only when low concentration of poly(I:C) (such as 4 ng/mL) is present, 4 based on below protocol with a minor modification by transfection again with low concentration of poly(I:C). Seed EPC cells (each well containing 0.5 mL M199 supplemented with 10% FBS) in 24-wells plates to reach monolayers after overnight (8–14 h) culture at 28°C.…”

“…Transfection of fish cells with poly(I:C), a synthetic analogue of virus dsRNA, is believed to mimic virus infection. The concentrations of poly(I:C) correspond to cellular concentrations of virus dsRNA produced during virus infection: low concentrations of transfected poly(I:C) might be analogue to the intracellular concentrations of viral dsRNA at the early stage of virus infection and high concentrations correspond to the late stage of virus infection 4 ( Figure 3 ). Seed EPC cells in 24-wells plates.…”

“…Given the conservation and disparities of zebrafish and human LGP2, this protocol can be used in cultured mammalian cells, including HEK293T and COS7 cells, with a minor modification. 4 …”

“…This protocol is designed to characterize the dual regulatory roles of zebrafish LGP2 in IFN response. 1 , 4 Comparison of WT and knockout zebrafish in response to SVCV infection demonstrates that LGP2 is essential for zebrafish survival against viral infection ( Figure 1 ). Real-time PCR analyses of ifn and ISG expression indicate that LGP2 acts as an activator of IFN response in the early phase of SVCV infection (<24 h post infection) and an inhibitor in the late phase of SVCV infection (>24 h post infection) ( Figure 2 ).…”