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, 6 (9), e24773

The Shrimp NF-κB Pathway Is Activated by White Spot Syndrome Virus (WSSV) 449 to Facilitate the Expression of WSSV069 (ie1), WSSV303 and WSSV371

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The Shrimp NF-κB Pathway Is Activated by White Spot Syndrome Virus (WSSV) 449 to Facilitate the Expression of WSSV069 (ie1), WSSV303 and WSSV371

Pei-Hui Wang et al. PLoS One.

Abstract

The Toll-like receptor (TLR)-mediated NF-κB pathway is essential for defending against viruses in insects and mammals. Viruses also develop strategies to utilize this pathway to benefit their infection and replication in mammal hosts. In invertebrates, the TLR-mediated NF-κB pathway has only been well-studied in insects and has been demonstrated to be important in antiviral responses. However, there are few reports of interactions between viruses and the TLR-mediated NF-κB pathway in invertebrate hosts. Here, we studied Litopenaeus vannamei Pelle, which is the central regulator of the Toll pathway, and proposed that a similar TLR/MyD88/Tube/Pelle/TRAF6/NF-κB cascade may exist in shrimp for immune gene regulation. After performing genome-wild analysis of white spot syndrome virus (WSSV) encoded proteins, we found that WSSV449 shows 15.7-19.4% identity to Tube, which is an important component of the insect Toll pathway. We further found that WSSV449 activated promoters of Toll pathway-controlled antimicrobial peptide genes, indicating WSSV449 has a similar function to host Tube in activating the NF-κB pathway. We suspected that WSSV449 activated the Toll-mediated NF-κB pathway for regulating viral gene expression. To test this hypothesis, we analyzed the promoters of viral genes and found 40 promoters that possess NF-κB binding sites. A promoter screen showed that the promoter activities of WSSV069 (ie1), WSSV303 and WSSV371 can be highly induced by the shrimp NF-κB family protein LvDorsal. WSSV449 also induced these three viral promoter activities by activating the NF-κB pathway. To our knowledge, this is the first report of a virus that encodes a protein similar to the Toll pathway component Tube to upregulate gene expression in the invertebrate host.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. cDNA sequences and genomic structure of LvPelle.
(A) The nucleotide (upper row) and deduced amino acid (lower row) sequences of the ORF are shown. The death domain (amino acids 14–69), and the protein kinase domain (amino acids 421–563) are shaded. (B) The genomic organization of LvPelle. The exons are depicted as boxes and introns as lines.
Figure 2
Figure 2. Domain architecture, phylogenetic tree and homology model of LvPelle.
(A) The schematic representation of the domain topology of LvPelle. LvPelle contains an organization that is typical of IRAK family proteins: N-terminal death domain and C-terminal protein kinase domain. (B) The phylogenetic tree of LvPelle with other IRAK family proteins. The numbers at the nodes indicate bootstrap values. LvPelle is boxed. AgPelle, Anopheles gambiae Pelle (Accession no. XP_311931); AmPelle, Apis mellifera Pelle (Accession no. XP_624002); CePelle, Caenorhabditis elegans Pelle/IL-1 receptor associated Kinase (IRAK) family member (pik-1) (Accession no. NP_502587); CqPelle, Culex quinquefasciatus Pelle (Accession no. EDS41908); DmPelle, Drosophila melanogaster Pelle (Accession no. NP_476971); TcPelle, Tribolium castaneum Pelle (Accession no. XP_966383); BfIRAK4, Branchiostoma floridae IRAK4 (Accession no. XP_002601719); BtIRAK4, Bos taurus IRAK4 (Accession no. NP_001069466); CiIRAK4, Ciona intestinalis IRAK4 (Accession no. XP_002122012); DrIRAK4, Danio rerio IRAK4 (Accession no. NP_956457); EsIRAK4, Euprymna scolopes IRAK4 (Accession no. AAY27972); GgIRAK4, Gallus gallus IRAK4 (Accession no. NP_001025909); HsIRAK4, Homo sapiens IRAK 4 (Accession no. NP_001107654); MmIRAK4, Mus musculus IRAK4 (Accession no. NP_084202); OmIRAK4, Oncorhynchus mykiss IRAK4 (Accession no. CBI63176); RnIRAK4, Rattus norvegicus IRAK4 (Accession no. XP_217026); TgIRAK4, Taeniopygia guttata IRAK4 (Accession no. XP_002194205); XtIRAK4, Xenopus tropicalis IRAK4 (Accession no. NP_001116877); BtIRAK1, B. taurus IRAK1 (Accession no. NP_001035645); DrIRAK1, D. rerio IRAK1 (Accession no. XP_697688); HsIRAK1, H. sapiens IRAK1 (Accession no. AAH54000); MmIRAK1, M. musculus IRAK1 (Accession no. NP_032389); TnIRAK1, Tetraodon nigroviridis IRAK1 (Accession no. CAF93411); XtIRAK1, X. tropicalis IRAK1 (Accession no. AAH75439); BtIRAK3, B. taurus IRAK3 (Accession no. NP_001177228); DrIRAK3, D. rerio IRAK3 (Accession no. AAH98615); HsIRAK3, H. sapiens IRAK3 (Accession no. NP_009130); MmIRAK3, M. musculus IRAK3 (Accession no. AAM83393); RnIRAK3, R. norvegicus IRAK3 (Accession no. NP_001101571); BtIRAK2, B. taurus IRAK2 (Accession no. NP_001069164); GgIRAK2, G. gallus IRAK2 (Accession no. NP_001025776); HsIRAK2, H. sapiens IRAK2 (Accession no. NP_001561); RnIRAK2, R. norvegicus IRAK2 (Accession no. AAH98060); TgIRAK2, T. guttata IRAK2 (Accession no. XP_002187461); XlIRAK2, Xenopus laevis IRAK2 (Accession no. NP_001079489). (C) Primary sequence alignments and homology models of the death domain and protein kinase domain of LvPelle. The death domain of LvPelle shows 21.2% identity to both Drosophila melanogaster and Mus musculus. The protein kinase domain of LvPelle shows 35.1% and 42.6% identity with Drosophila melanogaster and Homo sapiens, respectively. Homology models of the LvPelle death domain (b) and kinase domain (d) show high similarities with the crystal structures of Drosophila Pelle (a) and mammalian IRAK4 (c), respectively, providing the foundations of the evolutionarily conserved function of NF-κB signaling for LvPelle.
Figure 3
Figure 3. Expression of LvPelle in healthy and immune challenged shrimp.
(A) The tissue distribution of LvPelle in healthy shrimp by qPCR analysis. The expression of LvPelle in the pyloric caecum was set to 1.0. The temporal expression of LvPelle in the gill (B), intestine (C) and hepatopancreas (D) after V. alginolyticus and WSSV challenge. The expression values were normalized to LvEF-1α expression values using the relative standard curve method. qPCR was conducted in three replicates per sample. Data are expressed as the mean fold change (mean ± S.E., n = 3) from the untreated group. Statistical significance was calculated by Tukey multiple comparison tests and Student's t-test. The bars with different letters indicate statistical differences (p<0.05).
Figure 4
Figure 4. The functional study of LvPelle in the Toll pathway.
(A) The intracellular localization of LvPelle and its truncated mutants fused with GFP in S2 cells. P1 represented amino acids 1–69 of LvPelle, P2 represented amino acids 1–262 of LvPelle, P3 represented amino acids 1–536 of LvPelle (the full-length protein of LvPelle) and P4 represented amino acids 129–536 of LvPelle as indicated in Fig. 2. (B) Overexpression of LvPelle activates Drosophila and shrimp AMP promoters. Luciferase reporter genes including pGL3-PEN453, pGL3-PEN309, pGL3-PEN4, pGL3-Drs and pGL3-AttA were constructed successfully and demonstrated to be predominantly regulated through NF-κB activation , , , , , . In this study, we use these luciferase reporter genes to investigate the activation of Toll-mediated NF-κB pathway. The data are representative of three independent experiments. **p<0.01. (C) LvPelle associates with LvTRAF6 during TLR signal transduction. Myc-tagged LvPelle co-precipitated with V5-tagged LvTRAF6 (lane 1) and V5-tagged LvPelle coprecipitated with Myc-tagged LvTRAF6 (lane 3). pAc5.1-Basic were used as controls.
Figure 5
Figure 5. WSSV449 shows similarity to host Tube and activates the promoters of Drosophila and shrimp AMPs.
(A) Multiple sequence alignment of WSSV449 and insect Tube proteins. The conserved functional death domain is framed with a green line. The overall protein identities are indicated. WSSV449 shows 15.7-19.4% identity to Tube, which is a positive regulator of insect Toll pathway. CfTube, Camponotus floridanus (Accession no. EFN72687); DmTube, Drosophila melanogaster (Accession no. AAA28994); DpTube, Drosophila persimilis (Accession no. EDW34648); HsTube, Harpegnathos saltator (Accession no. EFN87560); WSSV449, white spot syndrome virus 449 (Accession no. AAL89317). (B) Overexpression of WSSV449 activates Drosophila and shrimp AMP promoters. The data are representative of three independent experiments. **p<0.01.
Figure 6
Figure 6. A promoter screen to identify viral genes induced by NF-κB activation.
(A) The determination of the promoter activities of 40 WSSV genes when the shrimp NF-κB family protein LvDorsal is overexpressed in S2 cells. All of the 40 WSSV genes possess NF-κB binding sites in their promoter regions. The promoter regions were inserted into pGL3-Basic to construct luciferase reporters. When transfected into S2 cells, the promoters of WSSV069 (ie1), WSSV303 and WSSV371 are activated by LvDorsal. (B) Stimulated by WSSV, LvDorsal translocated to the nucleus. PBS treated cells were used as a control. (C) The promoter regions containing NF-κB binding sites of WSSV069 (ie1), WSSV303 and WSSV371. The NF-κB binding sites were in bold and underlined.
Figure 7
Figure 7. WSSV449 and LvPelle activate the promoters of WSSV069 (ie1), WSSV303 and WSSV371.
The S2 cells were transfected with 0.05 µg of protein expression vector (pAC5.1-LvPelle or pAC5.1-WSSV449), with 0.05 µg reporter gene plasmid (pGL3-Bsic, pGL3-WSSV069, pGL3-WSSV303 or pGL3-WSSV371) and with 0.005 µg pRL-TK Renilla luciferase plasmid (as an internal control, Promega, USA). Thirty-six hours after transfection, the cells were harvested and analyzed using the Dual Luciferase kit. The data are representative of three independent experiments. **p<0.01.

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