Junfeng Xie

Two Litopenaeus vannamei HMGB proteins interact with transcription factors LvSTAT and LvDorsal to activate the promoter of white spot syndrome virus immediate-early gene ie1.

White spot syndrome virus (WSSV) has caused great economic damage to shrimp aquaculture. Previous studies have shown that WSSV successfully usurps the immunity system of the host for its own gene regulation. To investigate the role of shrimp high mobility group box (HMGB) proteins in WSSV gene regulation, two Litopenaeus vannamei HMGB genes, LvHMGBa and LvHMGBb, were isolated by rapid amplification of cDNA ends (RACE). Recombinant LvHMGBa/b proteins were present in the nucleus of transfected Drosophila Schneider 2 (S2) cells. Luciferase reporter assays revealed that LvHMGBa/b upregulated the WSSV immediate-early (IE) gene (ie1) in a NF-κB and STAT binding site-dependent manner. GST pull-down assays demonstrated that LvHMGBa/b interacted with L. vannamei Dorsal (LvDorsal) and L. vannamei STAT (LvSTAT), respectively. LvHMGBa was highly expressed in hepatopancreas while HMGBb was highly expressed in stomach, intestine, heart, antennal gland, and epidermis. Moreover, an immune challenge assay demonstrated that the expression of LvHMGBa/b was upregulated by WSSV infection and that both mRNAs reached peak values at 24 h post-infection. To our knowledge, this is the first report that invertebrate HMGB proteins participates in viral gene regulation.

The Viral TRAF Protein (ORF111L) from Infectious Spleen and Kidney Necrosis Virus Interacts with TRADD and Induces Caspase 8-mediated Apoptosis

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus of the Iridoviridae family. It causes a serious and potentially pandemic disease in wild and cultured fishes. ISKNV infection induces evident apoptosis in mandarin fish (Siniperca chuatsi) and zebrafish (Danio renio). However, the mechanism is still unknown. After a genome-wide bioinformatics analysis of ISKNV-encoded proteins, the ISKNV open reading frame 111L (ORF111L) shows a high similarity to the tumour necrosis factor receptor-associated factor (TRAF) encoded by fish, mice and mammals, which is essential for apoptotic signal transduction. Moreover, ORF111L was verified to directly interact with the zebrafish TNF receptor type 1 associated death domain protein (TRADD). A recombinant plasmid containing the DNA sequence of ORF111L was constructed and microinjected into zebrafish embryos at the 1–2 cell stage to investigate its biological function in vivo. ORF111L overexpression in the embryos resulted in increased apoptosis. ORF111L-induced apoptosis was clearly associated with significant caspase 8 upregulation and activation. The knockdown of zebrafish caspase 8 expression effectively blocked the apoptosis induced by ORF111L overexpression. Significantly, ORF111L overexpression resulted in much stronger effect on caspase 8 and caspase 3 upregulation compared to zebrafish TRAF2. This is the first report of a viral protein similar to TRAF that interacts with TRADD and induces caspase 8-mediated apoptosis, which may provide novel insights into the pathogenesis of ISKNV infection.

Identification of two novel membrane proteins from the Tiger frog virus (TFV).

The Tiger frog virus (TFV) belongs to the genus Ranavirus in the family Iridoviridae, and its genome was completely sequenced in 2002. In order to better understand the viral structure and functional genes involved in infection and virus-host interactions, two candidate genes, ORF001L and ORF020R, were selected for our study. ORF001L and ORF020R were analyzed by genomic comparison and by using the TMHMM software. Both genes were conserved in the genus Ranavirus, may encode putative membrane proteins, and were determined as late genes by temporal analysis. In order to identify whether these two proteins were structural proteins or not, ORF001L and ORF020R were cloned and expressed in the pET32a (+) vector. Antisera against the two proteins were prepared by immunization of mice with purified proteins. Western blot analysis suggested that both ORF001L and ORF020R were structural proteins. Indirect immunofluorescence assay (IFA) revealed that the subcellular location of the two proteins was confined to the cytoplasm, especially at the viral assembly site (AS). Immunogold electron microscopy (IEM) further localized these two proteins, showing that they were envelope proteins.

Genome-wide analyses of proliferation-important genes of Iridovirus-tiger frog virus by RNAi

Tiger frog virus (TFV), a species of genus Ranavirus in the family Iridoviridae, is a nuclear cytoplasmic large DNA virus that infects aquatic vertebrates such as tiger frog (Rana tigrina rugulosa) and Chinese soft-shelled turtle (Trionyx sinensis). Based on the available genome sequences of TFV, the well-developed RNA interference (RNAi) technique, and the reliable cell line for infection model, we decided to analyze the functional importance of all predicted genes. Firstly, a relative quantitative cytopathogenic effect (Q-CPE) assay was established to monitor the viral proliferation in fish cells. Then, genome-wide RNAi screens of 95 small interference (si) RNAs against TFV were performed to characterize the functional importance of nearly all (95%) predicted TFV genes by Q-CPE scaling system. We identified 32 (33.7%) genes as essential, 50 (52.6%) genes as semi-essential and 13 (13.7%) genes as nonessential for TFV proliferation. Quantitative RT-PCR and titer assays of selected genes were performed to verify the screen results. Furthermore, the screened essential genes were analyzed for their genome distribution and conservative comparison within Ranavirus. Such a systematic screen for viral functional genes by cell phenotypes should provide further insights into understanding of the information in antiviral targets, and in viral replication and pathogenesis of iridovirus.

The core ubiquitin system of mandarin fish, Siniperca chuatsi, can be utilized by infectious spleen and kidney necrosis virus

ABSTRACT The process of ubiquitination regulates various cellular processes. The ubiquitin‐proteasome system (UPS) in fish, which is important for the generation of innate and adaptive immune responses to pathogens, is the target of aquatic viruses to achieve immune evasion. We cloned and characterized three genes, namely, a ubiquitin‐activating enzyme (ScE1), a ubiquitin‐conjugating enzyme (ScE2), and a HECT‐type ubiquitin ligase (ScE3) of mandarin fish Siniperca chuatsi. The genes were expressed in all tissues and the highest levels were observed in the blood. In infectious spleen and kidney necrosis virus (ISKNV)‐infected mandarin fish fry cells, the expression levels of the three genes in vitro were almost identical, and upregulated during the early stage and downregulated at the late stage. In the blood of ISKNV‐infected mandarin fish, their expressions in vivo were downregulated equally although peaking at different timepoints, indicating the suppression of UPS by viral infection. Furthermore, these recombinant proteins were determined to function well in ubiquitination assays in vitro. Moreover, ScE1 and ScE2 can be utilized by four RING‐type viral E3s (vE3s) that are encoded by ISKNV. The in vitro activity of vE3 was stronger than that of ScE3, suggesting that the fish UPS may be hijacked by ISKNV via E3 activity competition and expression modulation. The present study investigated the function of mandarin fish UPS as well as its response to iridovirus infection, providing insights to better understand the virus‐host interactions and the mechanism of ISKNV in evading host immune responses. HighlightsFull‐length cDNAs of ScE1, ScE2, and ScE3 were cloned and characterized.Their expression patterns were identified in healthy and infected cells and fish.ScE1, ScE2, and ScE3 functioned well in ubiquitination assays in vitro.ScE1 and ScE2 can be utilized by viral E3s encoded by ISKNV.The E3 activity of VP66 was stronger than ScE3 to achieve the hijack of host UPS.

The complete mitochondrial genome of the Epinephelus corallicola (Perciformes: Serranidae)

Abstract We presented the complete mitochondrial genome of the Epinephelus corallicola in this study. The mitochondrial genome is 16,647 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The gene order and composition of Epinephelus corallicola mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 26.60% of A, 15.89% of C, 28.66% of T and 28.85% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand.

Protein A from orange-spotted grouper nervous necrosis virus triggers type I interferon production in fish cell

ABSTRACT Family Nodaviridae consists of two genera: Alphanodavirus and Betanodavirus, and the latter is classified into four genotypes, including red‐spotted grouper nervous necrosis virus, tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, and barfin flounder nervous necrosis virus. Type I interferons (IFNs) play a central role in the innate immune system and antiviral responses, and the interactions between IFN and NNV have been investigated in this study. We have found that the RNA‐dependent RNA polymerase (RdRp) from orange‐spotted nervous necrosis virus (OGNNV), named protein A, was capable of activating IFN promoter in fathead minnow (FHM) cells. Transient expression of protein A was found to induce IFN expression and secretion, endowing FHM cells with anti‐tiger frog virus ability. Protein A from SJNNV can also induce IFN expression in FHM cells but that from Flock House virus (FHV), a well‐studied representative species of genus Alphanodavirus, cannot. RdRp activity and mitochondrial localization were shown to be required for protein A to induce IFN expression by means of activating IRF3 but not NF&kgr;B. Furthermore, DsRNA synthesized in vitro transcription and poly I:C activated IFN promoter activity when transfected into FHM cells, and dsRNA were also detected in NNV‐infected cells. We postulated that dsRNA, a PAMP, was produced by protein A, leading to activation of innate immune response. These results suggest that protein As from NNV are the agonists of innate immune response. This is the first work to demonstrate the interaction between NNV protein A and innate immune system, and may help to understand pathogenesis of NNV. HIGHLIGHTSThe IFN‐induced function of OGNNV protein A was identified.RdRp activity and mitochondrial localization are required for the IFN‐induced function of OGNNV protein A.Protein A from OGNNV and SJNNV but not from FHV can induce IFN expression.IRF3 is essential to protein A‐mediated IFN induction.IFN induced by protein A is sufficient to inhibit virus replication.

Prokaryotic Production of Virus-Like Particle Vaccine of Betanodavirus.

Betanodaviruses are the causative agents of viral nervous necrosis (VNN), a serious disease of cultured marine fish worldwide. To control this disease, vaccines of subunit capsid proteins (recombinant proteins or peptides), inactivated viruses, and virus-like particles (VLPs) were developed. VLP, which is highly similar to the wild-type virus in virion structure and contains no viral genome, was proved as one of the good and safe vaccines that can activate humoral immune response in the long term and induce cellular and innate immunities in the early stage post-immunization. The VLP vaccines can be expressed in vitro either by Baculovirus-based or yeast-based eukaryotic system or by bacterial expression system. In this chapter, the prokaryotic expression and the subsequent purification of VLP of betanodavirus orange-spotted grouper nervous necrosis virus (OGNNV) are presented.