Fig 1.
The domain composition of LvNLRC.
(A) Schematic diagram of the predicted domains of LvNLRC. There are three predicted regions in LvNLRC, including an N-terminal region of unknown function (M1-P349), a central NACHT domain (G350-R750), and the C-terminal LRRs domain in series (L891-L1485). The C-terminal LRRs domain has a typical horseshoe structure. (B) The predicted 3D structure of LvNLRC. (C-D) The putative ligand-binding sites (the pink and blue position) in central NACHT domain (C) and the horseshoe LRRs domain (D).
Fig 2.
Phylogenetic analysis of LvNLRC based on its full-length amino acid sequence.
Amino acid sequences of NLRs or NLR-like genes from different species, including Homo sapiens, Daphnia pulex, Eurytemora affinis, Hyalella Azteca, Armadillidium nasatum, Chionoecetes opilio, Portunus trituberculatus, Homarus americanus, Trachymyrmex cornetzi, Acromyrmex insinuator, Acromyrmex heyeri, Acromyrmex echinatior, Acromyrmex charruanus, Cyphomyrmex costatus, Apostichopus japonicus, Actinia tenebrosa, Exaiptasia diaphana, Stylophora pistillata, and Nematostella vectensis, were obtained from GenBank or Uniprot databases. The GenBank or Uniprot accession number of each sequence was shown in each branch. The numbers on the phylogenetic tree represent the bootstrap value.
Fig 3.
Tissue distribution and the responses of LvNLRC to WSSV infection.
(A) The Tissue distribution of LvNLRC. Ordinary one-way ANOVA was performed for statistical analysis. (B-C) Expression changes of LvNLRC at different time points after WSSV infection in Oka (B) and intestine (C). (D) The interference efficiency of dsLvNLRC in intestine, Oka and hemocytes. (E) The WSSV copies of LvNLRC-knockdown shrimp at 24 hpi and 48 hpi. dsEGFP, the control group injected with EGFP dsRNA and infected with WSSV. dsLvNLRC, the experimental group injected with LvNLRC dsRNA and infected with WSSV. Abbreviation interpretation in (A), Hp, hepatopancreas; Ms, muscle; Epi, epithelial; In, intestine; Oka, lymphoid organ; St, stomach; Ht, heart; Hc, hemocytes; Es, eyestalk; Gi, gill; Bra, brain; Vn, ventral nerve cord; Tg, thoracic ganglia. Two-way ANOVA was performed for statistical analysis of (B)(C)(D)(E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 4.
Molecular mechanism of LvNLRC in antiviral immune response.
(A) The binding ability of rLRRs of LvNLRC to poly(dA:dT) detected by ELISA. (B) The binding ability of rLRRs of LvNLRC to dsDNA encoding partial VP24 of WSSV. The assay was detected by ELISA. (C) The SPR (surface-plasmon resonance) kinetic fitting of 32a-LvNLRC-LRRs protein binding to poly(dA:dT). The interaction dynamics between 32a-LvNLRC-LRRs and poly(dA:dT) across a concentration gradient from 0.03125 μM to 1 μM. The molecular weight of poly(dA:dT) is calculated according to the molecular weight of the monomer (dA + dT), because there is no fixed molecular weight of poly(dA:dT). (D) The negative rTrx control expressed by the empty pET-32a-vector plasmid of the SPR assay across a higher concentration gradient from 3.125 μM to 100 μM. (E) Co-immunoprecipitation analysis of the homo-dimerization of LvNLRC. (F) Co-immunoprecipitation analysis of the interaction between the NACHT domain and LRRs domain of LvNLRC. (G) Co-immunoprecipitation analysis of the interaction between the LvNLRC-NACHT domain and LvSTING. (H) The effect of poly(dA:dT) on the association between LvNLRCNACHT-LRR and LvSTING. (I) Effect of LvNLRC knockdown on LvVago5 expression level in Oka. 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.
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;.
Fig 6.
The effect of poly(dA:dT) on the association between LvCypA and LvSTING.
(A) The interaction between LvCypA and LvNLRCNACHT-LRR. (B) Effects of different doses of poly(dA:dT) on the interaction between LvCypA and LvSTING, LvNLRC and LvSTING.
Fig 7.
Working model for the LvNLRC-mediated WSSV recognition and activation of Vago5 expression.
Normally in healthy shrimp, LvNLRC functions as a switch in antiviral immune signaling pathways rather than an immune escape tool for WSSV. Its NACHT domain competitively binds to STING, inhibiting the transduction of antiviral signals. LvCypA is also attached to the NACHT domain, and their interaction may inhibit the ATPase activity of NACHT domain, thus impeding LvSTING release and downstream antiviral signaling pathway activation. Upon WSSV infection, some viral particles enter the autophagy-lysosome pathway and the viral genome DNA might be released after autolysosome degradation [39]. The LRRs domain of LvNLRC can recognize the viral DNA nucleic acid. This recognition might trigger LvCypA activity. Consequently, LvCypA dissociates from the NACHT domain, which leads to the dissociation of LvSTING. This activates the downstream IRF-Vago signaling pathway, increasing the production of LvVago5 to participate in host antiviral immunity.