An invertebrate NLR recognizes viral nucleic acids and balances the antiviral signaling pathway through interaction with STING and Cyclophilin A
Fig 5
Screening of LvNLRC interacting molecule and its immune responses during WSSV infection in vivo.
(A) The interaction of LvCypA with full-length LvNLRC verified by yeast two-hybrid. Line1, self-activation test group, Y2H [PGBKT7-A+ PGADT7]. Line 2, experimental group, Y2H [PGBKT7-A+ PGADT7-B]. Line 3, positive control group, Y2H [pGBKT7-53 + pGADT7-T]. Line 4, negative control group, Y2H [pGBKT7-lam + pGADT7-T]. The vertical axis represents the dilution ratio of the spot strain. (B) Co-immunoprecipitation of LvCypA with the three domains (N-terminal, NACHT and LRRs) of LvNLRC. LvNLRCN-end is the N-terminal domain of LvNLRC. LvNLRCNACHT is the NACHT domain of LvNLRC. LvNLRCLRR is the LRRs domain of LvNLRC. Anti-flag-coupled magnetic beads were used for co-immunoprecipitation to detect V5 tagged prey proteins in Co-IP samples by Anti-V5 antibodies. WCL, whole cell lysate. The Flag tagged protein carries His tag and is detected with anti-His antibodies. (C) Knockdown of LvCypA by dsRNA. Unpaired t-test was performed for statistical analysis. (D) The WSSV copies of LvCypA-knockdown shrimp at 27 hpi and 48 hpi. dsEGFP, the control group injected with EGFP dsRNA and infected with WSSV. dsLvCypA, the experimental group injected with LvCypA dsRNA and infected with WSSV. In each treatment, the horizontal line represents the median quartile distance and the dot represents the number of WSSV copies (copies/ng DNA) in each parallel. Two-way ANOVA was performed for statistical analysis. (E) Effect of LvCypA knockdown on LvVago5 expression level in Oka. Unpaired t-test was performed for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;.