VP39 of Spodoptera litura multicapsid nucleopolyhedrovirus cannot efficiently rescue the nucleocapsid assembly of vp39-null Autographa californica multiple nucleopolyhedrovirus.

BACKGROUND: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vp39 is conserved in all sequenced baculovirus genomes. In previous studies, VP39 has been identified as the major capsid structure protein of baculoviruses and found to be essential for nucleocapsid assembly. The nucleocapsid composition and structure of Group I and II NPVs of the Alphabaculovirus genus are very similar. It is not clear whether the major capsid structure protein VP39 of Group I NPVs is functionally identical to or substitutable with the Group II NPV VP39. In this study, the function of Group II Spodoptera litura MNPV (SpltMNPV) VP39 in Group I AcMNPV was characterized. METHODS: Sequence alignment of AcMNPV VP39 and SpltMNPV VP39 was performed using Clustal X and edited with GeneDoc. To determine whether VP39 of Group I NPVs can be functionally substituted by Group II NPV VP39, a vp39-null AcMNPV (vAcvp39KO) and a vp39-pseudotyped AcMNPV (vAcSpltvp39:FLAG), in which the Group I AcMNPV vp39 coding sequence was replaced with that of SpltMNPV from Group II NPVs, were constructed via homologous recombination in Escherichia coli. Using an anti-FLAG monoclonal antibody, immunoblot analysis was performed to examine SpltMNPV VP39 expression. Fluorescence and light microscopy were used to monitor viral replication and infection. Viral growth curve analysis was performed using a fifty percent tissue culture infective dose (TCID50) endpoint dilution assay. Viral morphogenesis was detected using an electron microscope. RESULTS: Sequence alignment indicated that the N-termini of AcMNPV VP39 and SpltMNPV VP39 are relatively conserved, whereas the C-terminus of SpltMNPV VP39 lacks the domain of amino acid residues 306-334 homologous to AcMNPV VP39. Immunoblot analysis showed that SpltMNPV VP39 was expressed in vAcSpltvp39:FLAG. Fluorescence and light microscopy showed that vAcSpltvp39:FLAG did not spread by infection. Viral growth curve analysis confirmed a defect in infectious budded virion production. Electron microscopy revealed that although masses of abnormally elongated empty capsid structures existed inside the nuclei of Sf9 cells transfected with vAcSpltvp39:FLAG, no nucleocapsids were observed. CONCLUSION: Altogether, our results demonstrated that VP39 from SpltMNPV cannot efficiently substitute AcMNPV VP39 during nucleocapsid assembly in AcMNPV.

TRAF3 of blunt snout bream participates in host innate immune response to pathogenic bacteria via NF-κB signaling pathway.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a multifunctional adaptor protein primarily involved in both bacterial defense and antiviral immunity in living organisms. However, the knowledge on TRAF3 in blunt snout bream (Megalobrama amblycephala), a freshwater fish with economic values, remained unclear. In the present study, we identified and characterized successfully Traf3 gene from M. amblycephala (maTraf3). The maTraf3 cDNA contained a 1722 bp open reading frame that encoded a protein of 573 amino acid residues. The deduced amino acid sequence comprised of a RING finger domain, two zinc finger motifs, a coiled-coil region and a MATH domain. Analysis of the transcriptional patterns of maTraf3 revealed that it was ubiquitously distributed in various tissues tested from M. amblycephala, with the abundance of expression in spleen and muscle. Following a challenge with Aeromonas hydrophila and lipopolysaccharide stimulation, the expression of maTraf3 was strongly enhanced at different time points in vitro and in vivo. MaTRAF3 was identified as a cytosolic protein and suggested to form aggregates or be associated with vesicles scattering in the cytoplasm. NF-κB transcription was activated by maTraf3 in reporter assay. The overexpression of maTraf3 produced high levels of pro-inflammatory cytokines such as IL-1ß, IL-6, IL-8 and TNF-α, implying its immune-regulatory role in M. amblycephala. Taken together, our results obtained in this study demonstrated the crucial role of maTraf3 in mediating host innate immune response to pathogen invasion via NF-κB signaling pathway, which might indicate a novel therapeutic approach to combat bacterial infection in fish.

Identification and Characterization of Lipopolysaccharide Induced TNFα Factor from Blunt Snout Bream, Megalobrama amblycephala.

Lipopolysaccharide induced TNFα factor (LITAF) is an important transcription factor responsible for regulation of tumor necrosis factor α. In this study, a novel litaf gene (designated as Malitaf) was identified and characterized from blunt snout bream, Megalobrama amblycephala. The full-length cDNA of Malitaf was of 956 bp, encoding a polypeptide of 161 amino acids with high similarity to other known LITAFs. A phylogenetic tree also showed that Malitaf significantly clustered with those of other teleost, indicating that Malitaf was a new member of fish LITAF family. The putative maLITAF protein possessed a highly conserved LITAF domain with two CXXC motifs. The mRNA transcripts of Malitaf were detected in all examined tissues of healthy M. amblycephala, including kidney, head kidney, muscle, liver, spleen, gill, and heart, and with the highest expression in immune organs: spleen and head kidney. The expression level of Malitaf in spleen was rapidly up-regulated and peaked (1.29-fold, p < 0.05) at 2 h after lipopolysaccharide (LPS) stimulation. Followed the stimulation of Malitaf, Matnfα transcriptional level was also transiently induced to a high level (51.74-fold, p < 0.001) at 4 h after LPS stimulation. Taken together, we have identified a putative fish LITAF ortholog, which was a constitutive and inducible immune response gene involved in M. amblycephala innate immunity during the course of a pathogenic infection.

Identification and characterization of a novel Toll-like receptor 4 homologue in blunt snout bream, Megalobrama amblycephala.

Toll-like receptors (TLRs) are central players in the innate immune system in response to a wide range of pathogen infection. Among various TLRs, TLR4 plays a key role in recognition of bacterial lipopolysaccharides (LPS). In the present study, we identified and characterized a novel TLR4 homologue (maTLR4b) in blunt snout bream (Megalobrama amblycephala) which was significantly distinct from previously reported M. amblycephala TLR4 (tentatively named maTLR4a). The results showed that the complete cDNA sequence of maTLR4b was 3261 bp with an open reading frame encoding a polypeptide of 820 amino acids, and that its genomic sequence was 3793 bp, which had 3 exons. Structurally, the deduced maTLR4b protein showed a typical TLR domain architecture, including a signal peptide, eight leucine-rich repeats (LRRs) in the extracellular region, a transmembrane domain, and a Toll-Interleukin 1 receptor (TIR) domain in the cytoplasmic region. Phylogenetic analysis revealed that all TLR4s from teleost fish formed a monophyletic clade. Both maTLR4a and maTLR4b were divided into two distinct branches, and showed the highest level of similarity with the grass carp TLR4.2 and TLR4.4 homologue, respectively. MaTLR4b was constitutively expressed in all healthy tissues tested although at different levels. After LPS stimulation, the expression levels were significantly up-regulated in spleen, and peaked at 4 h between maTLR4a and maTLR4b, but with a distinct and complementary expression patterns. Taken together, these results suggested that maTLR4b is indeed a functional homologue of TLR4 in other species, which may play vital role in innate immune.

Natural Resistance Associated Macrophage Protein Is Involved in Immune Response of Blunt Snout Bream, Megalobrama amblycephala.

The natural resistance-associated macrophage protein gene (Nramp), has been identified as one of the significant candidate genes responsible for modulating vertebrate natural resistance to intracellular pathogens. Here, we identified and characterized a new Nramp family member, named as maNramp, in the blunt snout bream. The full-length cDNA of maNramp consists of a 153 bp 5'UTR, a 1635 bp open reading frame encoding a protein with 544 amino acids, and a 1359 bp 3'UTR. The deduced protein (maNRAMP) possesses the typical structural features of NRAMP protein family, including 12 transmembrane domains, three N-linked glycosylation sites, and a conserved transport motif. Phylogenetic analysis revealed that maNRAMP shares the significant sequence consistency with other teleosts, and shows the higher sequence similarity to mammalian Nramp2 than Nramp1. It was found that maNramp expressed ubiquitously in all normal tissues tested, with the highest abundance in the spleen, followed by the head kidney and intestine, and less abundance in the muscle, gill, and kidney. After lipopolysaccharide (LPS) stimulation, the mRNA level of maNramp was rapidly up-regulated, which reached a peak level at 6 h. Altogether, these results indicated that maNramp might be related to fish innate immunity and similar to mammalian Nramp1 in function.