Chicken-Derived Pattern Recognition Receptor chLGP2 Inhibits the Replication and Proliferation of Infectious Bronchitis Virus

Abstract

The widespread nature and economic importance of Infectious bronchitis virus (IBV) and interactions between IBV and the host immune response remain poorly understood. Understanding the mechanism of virus recognition via innate immunity can help resist IBV invasion. Retinoic acid-induced gene I-like receptor (RLRs) recognize virus RNA in virus infection, and LGP2 is a member of RLRs. According to the current studies, LGP2 exhibited certain inhibition in the virus, and there is a lack of investigation for chicken's LGP2. It is important to figure out the role of chLGP2 in host immune recognition of IBV. Our results showed that chLGP2 inhibited the proliferation of IBV Beaudette in cells. Also, chLGP2 can identify and combine with IBV RNA. The domains of chLGP2 were separately expressed and inspired by related literature, and the chLGP2 K30A mutant was constructed. Our results suggested its structural integrity and the adenosine triphosphatase (ATPase) activity are critical for IBV inhibiting activity. chTRBP was selected after CO-IP and Mass spectrometry test. We found chTRBP and chLGP2 are the interacting partners and promote mutual expression. Our study showed that chTRBP could also suppress IBV infections via chLGP2, which provided a basis for future innate immunity research for IBV.

Keywords: IBV; IFN-β; chLGP2; chTRBP; innate immunity.

FIGURE 1

Expression of chLGP2 in HD11 and DF-1 cells and effect on IBV Beaudette strain. chLGP2 overexpression significantly inhibited IBV Beaudette replication in (A) HD11 and (B) DF-1 cells. Virus titers (TCID₅₀) at 36 h post-infection in cell supernatant of (C) HD11 and (D) DF-1 cells. Expressions of (E) chLGP2 or (F) empty plasmid in HD11 cells. Expression of (G) chLGP2 and (H) empty plasmid in DF-1 cells. Blue fluorescence represents the nucleus, and green fluorescence represents the transfected chLGP2. Data are the mean values from three independent experiments, and error bars denote mean ± SDM. *P < 0.05; ^** P < 0.01; ns means not significant.

FIGURE 2

The cell and virus-specific effect of chLGP2. Post-IBV Beaudette infection, chLGP2 overexpression significantly upregulated IFN-β expression in (A) HD11 cells but not in (B) DF-1 cells. (C) chLGP2 overexpression did not inhibit the replication of IBV M41 in DF-1 cells. (D) chLGP2 did not affect the IFN-β expression during IBV Beaudette infection in DF-1 cells. Data are the mean values of three independent experiments, and error bars denote mean ± SDM.

FIGURE 3

Effect of chLGP2 ATPase activity and individual domains on IBV RNA binding. Compared with the control, chLGP2 K30A inhibited the replication of IBV in both (A) HD11 and (B) DF-1 cells at 36 h post-infection. However, chLGP2 K30A did not affect the production of IFN-β in (C) HD11 and (D) DF-1 cells. The mutant decreased the titer of the IBV B strain, but the inhibitory effect was weaker than that of wild type in both (E) HD11 and (F) DF-1 cells. (G) The IBV RNA binding ability of different chLGP2 constructs was as follows: chLGP2 > K30A > CTD > Helicase. Data are the mean values of three independent experiments, and error bars denote mean ± SDM. * P < 0.05; ^** P < 0.01; ^**** P < 0.0001; ns means not significant.

FIGURE 4

The interaction between chLGP2 and chTRBP and the expression of chLGP2 domains. (A) Extracellular expression of chLGP2, Helicase domain, and CTD. (B) Schematic of different domains and the mutant K30A of LGP2. (C) GO enrichment analysis of chLGP2 bound proteins based on mass spectrometry results.

FIGURE 5

The IBV inhibitory roles of chLGP2 domains in different cells. Compared with the helicase domain, the CTD domain of chLGP2 showed a certain inhibitory effect on IBV both in (A) HD11 and (B) DF-1 cells. (C) In HD11 cells, the individual domains of chLGP2 did not change the level of IFN-β, while in (D) DF-1 cells, the domains and the full chLGP2 showed almost the same effect. The CTD domain of chLGP2 inhibited the virus titer of IBV to a certain extent, both in (E) HD11 and (F) DF-1 cells. Data are the mean values of three independent experiments, and error bars denote mean ± SDM. * P < 0.05; ^** P < 0.01; ^*** P < 0.001; ns means not significant.

FIGURE 6

Effects of chTRBP overexpression and co-expression of chTRBP and chLGP2 on IBV replication and expression of immune-related genes: (A) IBV; (B) IFN-β; (C) PKR; (D) IL-1β; (E) chLGP2; (F) chTRBP; (G) Bcl-2; (H) FasL; (I) Caspase3; (J) Caspase8; (K) Caspase9. Data are the mean values from three independent experiments, and error bars denote mean ± SDM.