Epidermal growth factor receptor promotes infectious spleen and kidney necrosis virus invasion via PI3K-Akt signaling pathway.

Infectious spleen and kidney necrosis virus disease (ISKNVD) caused significant economic losses to the fishery industry. Epidermal growth factor receptor (EGFR), phosphatidylinositide 3-kinase (PI3K) played an important role in ISKNV invasion. However, the molecular regulatory mechanisms among EGFR, PI3K-Akt, and ISKNV invasion are not clear. In this study, ISKNV infection rapidly induced EGFR activation. While, EGFR activation promoted virus entry, but EGFR inhibitors and specific RNA (siRNA) decreased virus invasion. The PI3K-Akt as downstream signalling of EGFR was activated upon ISKNV infection. Consistent with the trends of EGFR, Akt activation increased ISKNV entry into cells, Akt inhibition by specific inhibitor or siRNA decreased ISKNV invasion. Akt silencing combination with EGFR activation showed that EGFR activation regulation ISKNV invasion is required for activation of the Akt signalling pathway. Those data demonstrated that ISKNV-induced EGFR activation positively regulated virus invasion by PI3K-Akt pathway and provided a better understanding of the mechanism of EGFR-PI3K-Akt involved in ISKNV invasion.

Protective immunity induced by ankyrin repeat-containing protein-based DNA vaccine against rock bream iridovirus (RBIV) in rock bream (Oplegnathus fasciatus).

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (Oplegnathus fasciatus) and remains an unsolved problem in Korea aquaculture industry. In this study, we assessed the potential of ankyrin repeat (ANK)-containing proteins to induce protective immunity in RBIV-infected rock bream. Rock bream administered with ankyrin repeat-containing protein-based DNA vaccine (200 ng/fish) exhibited significant protection against at 4 and 8 weeks post vaccination to infected with 6.7 × 105 RBIV at 23°C; relative percent survival (RPS) of 60.04% and 40.1%, respectively. Furthermore, survivors from the first infection were strongly protected from RBIV (1.1 × 107) re-infection at 70 days post infection, as 100% RPS was observed and without clinical signs of RBIV diseases. Moreover, TLR3 (9.5-fold), TLR9 (5.2-fold), MyD88 (15.9-fold), Mx (55.5-fold), ISG15 (19.0-fold), PKR (24.2-fold), MHC class I (5.1-fold), perforin (6.5-fold), Fas (6.4-fold), Fas ligand (7.1-fold), caspase8 (5.0-fold), caspase9 (12.5-fold), and caspase3 (6.3-fold) responses were significantly elevated in the muscle (vaccine injection site) of ANK-based DNA vaccinated fish at 7 days post vaccination. However, inflammatory cytokines (IL1ß, IL8, and TNFα) were not enhanced in the vaccinated rock bream. Moreover, ANK gene may be a good candidate to detect RBIV infection or in revealing specific information to elucidate the pathogenic mechanisms underlying RBIV infection. In summary, ANK-based DNA vaccination in rock bream induced TLR- and IFN-mediated or apoptosis-related immune responses and suggest efficient preventive measures against RBIV.

Mandarin Fish (Siniperca chuatsi) p53 Regulates Glutaminolysis Induced by Virus via the p53/miR145-5p/c-Myc Pathway in Chinese Perch Brain Cells.

p53, as an important tumor suppressor protein, has recently been implicated in host antiviral defense. The present study found that the expression of mandarin fish (Siniperca chuatsi) p53 (Sc-p53) was negatively associated with infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV) proliferation as well as the expression of glutaminase 1 (GLS1) and glutaminolysis pathway-related enzymes glutamate dehydrogenase (GDH) and isocitrate dehydrogenase 2 (IDH2). This indicated that Sc-p53 inhibited the replication and proliferation of ISKNV and SCRV by negatively regulating the glutaminolysis pathway. Moreover, it was confirmed that miR145-5p could inhibit c-Myc expression by targeting the 3' untranslated region (UTR). Sc-p53 could bind to the miR145-5p promoter region to promote its expression and to further inhibit the expression of c-Myc. The expression of c-Myc was proved to be positively correlated with the expression of GLS1 as well. All these suggested a negative relationship between the Sc-p53/miR145-5p/c-Myc pathway and GLS1 expression and glutaminolysis. However, it was found that after ISKNV and SCRV infection, the expressions of Sc-p53, miR145-5p, c-Myc, and GLS1 were all significantly upregulated, which did not match the pattern in normal cells. Based on the results, it was suggested that ISKNV and SCRV infection altered the Sc-p53/miR145-5p/c-Myc pathway. All of above results will provide potential targets for the development of new therapeutic strategies against ISKNV and SCRV. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV) as major causative agents have caused a serious threat to the mandarin fish farming industry (J.-J. Tao, J.-F. Gui, and Q.-Y. Zhang, Aquaculture 262:1-9, 2007, https://doi.org/10.1016/j.aquaculture.2006.09.030). Viruses have evolved the strategy to shape host-cell metabolism for their replication (S. K. Thaker, J. Ch'ng, and H. R. Christofk, BMC Biol 17:59, 2019, https://doi.org/10.1186/s12915-019-0678-9). Our previous studies showed that ISKNV replication induced glutamine metabolism reprogramming and that glutaminolysis was required for efficient replication of ISKNV and SCRV. In the present study, the mechanistic link between the p53/miR145-5p/c-Myc pathway and glutaminolysis in the Chinese perch brain (CPB) cells was provided, which will provide novel insights into ISKNV and SCRV pathogenesis and antiviral treatment strategies.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Lymphocystis Disease Virus (Iridoviridae) Enters Flounder (Paralichthys olivaceus) Gill Cells via a Caveolae-Mediated Endocytosis Mechanism Facilitated by Viral Receptors.

In previous research, voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) in flounder (Paralichthys olivaceus) were confirmed as functional receptors for lymphocystis disease virus (LCDV) entry; however, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear. In this study, we elucidated the endocytosis pathway of LCDV entry into flounder gill (FG) cells by treatment with specific inhibitory agents, siRNAs, and co-localization analysis. LCDV entry was significantly inhibited by the disruption of caveolae-mediated endocytosis, dynamin, and microtubules, and the knockdown of caveoline-1 and dynamin expression, but was not inhibited by the disruption of clathrin-mediated endocytosis, micropinocytosis, or low-pH conditions. The disruption of caveolae-mediated and clathrin-mediated endocytosis was verified by the internalization of cholera toxin subunit B (CTB) and transferrin, respectively. Confocal immunofluorescence assay demonstrated that LCDV was co-localized with VDAC2 and RACK1, CTB was co-localized with VDAC2 and RACK1 and partially with LCDV, but transferrin was not co-localized with LCDV, VDAC2, or RACK1, indicating that LCDV utilized the same pathway as CTB, i.e., caveolae-mediated endocytosis. This was different from the pathway of transferrin, which used clathrin-mediated endocytosis. Furthermore, caveolin-1 was co-localized with LCDV, VDAC2, and RACK1, suggesting that caveolin-1 was involved in LCDV entry. These results revealed for the first time that LCDV entered into FG cells via caveolae-mediated endocytosis facilitated by VDAC2 and RACK1 receptors, relying on dynamin and microtubules in a pH-independent manner, which provided new insight into the molecular mechanisms of LCDV entry and potential for the development of antiviral agents, expanding our understanding of iridovirus infection.

Genetic and codon usage bias analyses of major capsid protein gene in Ranavirus.

The Ranavirus (one genus of Iridovidae family) is an emerging pathogen that infects fish, amphibian, and reptiles, and causes great economical loss and ecological threat to farmed and wild animals globally. The major capsid protein (MCP) has been used as genetic typing marker and as target to design vaccines. Herein, the codon usage pattern of 73 MCP genes of Ranavirus and Lymphocystivirus are studied by calculating effective number of codons (ENC), relative synonymous codon usage (RSCU), codon adaptation index (CAI), and relative codon deoptimization index (RCDI), and similarity index (SiD). The Ranavirus are confirmed to be classified into five groups by using phylogenetic analysis, and varied nucleotide compositions and hierarchical cluster analysis based on RSCU. The results revealed different codon usage patterns among Lymphocystivirus and five groups of Ranavirus. Ranavirus had six over-represented codons ended with G/C nucleotide, while Lymphocystivirus had six over-represented codons ended with A/T nucleotide. A comparative analysis of parameters that define virus and host relatedness in terms of codon usage were analyzed indicated that Amphibian-like ranaviruses (ALRVs) seem to possess lower ENC values and higher CAIs in contrast to other ranaviruses isolated from fishes, and two groups (FV3-like and CMTV-like group) of them had received higher selection pressure from their hosts as having higher relative codon deoptimization index (RCDI) and similarity index (SiD). The correspondence analysis (COA) and Spearman's rank correlation analyses revealed that nucleotide compositions, relative dinucleotide frequency, mutation pressure, and natural translational selection shape the codon usage pattern in MCP genes and the ENC-GC3S and neutrality plots indicated that the natural selection is the predominant factor. These results contribute to understanding the evolution of Ranavirus and their adaptions to their hosts.

Accelerated Metabolite Levels of Aerobic Glycolysis and the Pentose Phosphate Pathway Are Required for Efficient Replication of Infectious Spleen and Kidney Necrosis Virus in Chinese Perch Brain Cells.

Glucose is a main carbon and energy source for virus proliferation and is usually involved in the glycolysis, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA cycle) pathways. In this study, we investigated the roles of glucose-related metabolic pathways during the replication of infectious spleen and kidney necrosis virus (ISKNV), which has caused serious economic losses in the cultured Chinese perch (Siniperca chuatsi) industry. We found that ISKNV infection enhanced the metabolic pathways of the PPP and the TCA cycle at the early stage of the ISKNV infection cycle and enhanced the glycolysis pathway at the late stage of the ISKNV infection cycle though the comprehensive analysis of transcriptomics, proteomics, and metabolomics. The advanced results proved that ISKNV replication induced upregulation of aerobic glycolysis at the late stage of ISKNV infection cycle and aerobic glycolysis were required for ISKNV multiplication. In addition, the PPP, providing nucleotide biosynthesis, was also required for ISKNV multiplication. However, the TCA cycle involving glucose was not important and necessary for ISKNV multiplication. The results reported here provide new insights into viral pathogenesis mechanism of metabolic shift, as well as antiviral treatment strategies.

Voltage-Dependent Anion Channel Protein 2 (VDAC2) and Receptor of Activated Protein C Kinase 1 (RACK1) Act as Functional Receptors for Lymphocystis Disease Virus Infection.

In previous research, a 27.8-kDa protein in flounder Paralichthys olivaceus gill (FG) cells was identified as a putative cellular receptor (27.8R), which mediated lymphocystis disease virus (LCDV) infection via interaction with a 32-kDa viral attachment protein (VAP) of LCDV, and monoclonal antibodies (MAbs) against 27.8R and 32-kDa VAP were developed. In this study, the 27.8R was identified as voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1) of flounder. Recombinant VDAC2 (rVDAC2) and RACK1 (rRACK1) were obtained by prokaryotic expression, and rabbit anti-VDAC2/RACK1 polyclonal antibodies were prepared. The rVDAC2, rRACK1, and 27.8-kDa proteins in FG cells were recognized by anti-27.8R MAbs and anti-VDAC2/RACK1 polyclonal antibodies simultaneously. Preincubation of FG cells with anti-VDAC2/RACK1 polyclonal antibodies significantly decreased the percentages of LCDV-infected cells and LCDV copy numbers, blocked virus infection, and delayed the development of cytopathic effect. The mRNA expressions of VDAC2 and RACK1 in FG cells were upregulated to maximum levels 12 h and 48 h after LCDV infection, respectively. VDAC2/RACK1 knockdown through short interfering RNA (siRNA) significantly reduced VDAC2/RACK1 expression and LCDV copy numbers in FG cells compared with negative controls, while VDAC2/RACK1 expression on LCDV-nonpermissive epithelial papillosum cells (EPCs) conferred susceptibility to LCDV infection, indicating the VDAC2 and RACK1 were sufficient to allow LCDV entry and infection. All these results collectively showed that VDAC2 and RACK1 function as receptors for LCDV entry and infection.IMPORTANCE Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease in fish, which has caused huge economic losses to the aquaculture industry worldwide, but the molecular mechanism underlying the LCDV-host interaction remains unclear. Here, the 27.8-kDa putative cellular receptor for LCDV was identified as voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1), and our results revealed that VDAC2 and RACK1 expression was sufficient to allow LCDV entry and that they are functional receptors that initiate LCDV infection for the first time, which leads to a better understanding of the molecular mechanism underlying LCDV infection and virus-host interactions.

Characterization of a new member of Iridoviridae, Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp (Litopenaeus vannamei).

A newly discovered iridescent virus that causes severe disease and high mortality in farmed Litopenaeus vannamei in Zhejiang, China, has been verified and temporarily specified as shrimp hemocyte iridescent virus (SHIV). Histopathological examination revealed basophilic inclusions and pyknosis in hematopoietic tissue and hemocytes in gills, hepatopancreas, periopods and muscle. Using viral metagenomics sequencing, we obtained partial sequences annotated as potential iridoviridae. Phylogenetic analyses using amino acid sequences of major capsid protein (MCP) and ATPase revealed that it is a new iridescent virus but does not belong to the five known genera of Iridoviridae. Transmission electron microscopy showed that the virus exhibited a typical icosahedral structure with a mean diameter of 158.6 ± 12.5 nm (n = 30)(v-v) and 143.6 ± 10.8 nm (n = 30)(f-f), and an 85.8 ± 6.0 nm (n = 30) nucleoid. Challenge tests of L. vannamei via intermuscular injection, per os and reverse gavage all exhibited 100% cumulative mortality rates. The in situ hybridization showed that hemopoietic tissue, gills, and hepatopancreatic sinus were the positively reacting tissues. Additionally, a specific nested PCR assay was developed. PCR results revealed that L. vannamei, Fenneropenaeus chinensis, and Macrobrachium rosenbergii were SHIV-positive, indicating a new threat existing in the shrimp farming industry in China.

Molecular cloning and characterization of interferon regulatory factor 9 (IRF9) in Japanese flounder, Paralichthys olivaceus.

Interferon regulatory factor 9 (IRF9) in mammals is known to be involved in antiviral response. In this study, we studied the structure, mRNA tissue distribution and regulation of IRF9 from Japanese flounder, Paralichthys olivaceus. The cDNA sequence of IRF9 is 3305 bp long, containing an open reading frame (ORF) of 1308 bp that encodes a peptide of 435 amino acids. The predicted protein sequence shares 33.7-72.0% identity to other fish IRF9s. Japanese flounder IRF9 possesses a DNA-binding domain (DBD), an IRF association domain (IAD), two nuclear localization signals (NLSs) and a proline-rich domain (PRD). The IRF9 transcripts were detectable in all examined tissues of healthy Japanese flounders, with higher levels in the head kidney, kidney, liver and spleen. The IRF9 mRNA levels were up-regulated in the gills, head kidney, spleen and muscle when challenged with polyinosinic:polycytidylic acid (poly I:C) or lymphocystis disease virus (LCDV). The up-regulations were stronger and arose earlier in the case of poly I:C treatment in most tested organs in a 7-day time course, with maximum increases ranging from 1.37- to 8.59-fold and peak time points from 3 h to 3 d post injection depending on different organs, relative to those in the case of LCDV treatment which ranged from 1.32- to 3.21-fold and from 18 h to 3 d post injection, respectively. The highest and earliest inductions were detected in the spleen in both challenge cases, while the inductions by LCDV in the muscle were quite faint. These results demonstrate a role of Japanese flounder IRF9 in the host's antiviral responses.

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 a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection.

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase ß.

The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs, found predominantly in eukaryotes and bacteria, but the functions of the ANK repeat are rarely researched in animal viruses, with the exception of poxviruses. Infectious spleen and kidney necrosis virus (ISKNV) is a typical member of the genus Megalocytivirus in the family Iridoviridae and is a causative agent of epizootics in fish. The genome of ISKNV contains four putative viral ANK (vANK) repeat proteins and their functions remain largely unknown. In the present study, it was found that ORF124L, a vANK repeat protein in ISKNV, encodes a protein of 274 aa with three ANK repeats. Transcription of ORF124L was detected at 12 h post-infection (p.i.) and reached a peak at 40 h p.i. ORF124L was found to localize to both the nucleus and the cytoplasm in mandarin fish fry cells. ISKNV ORF124L interacted with the mandarin fish IκB kinase ß protein (scIKKß), and attenuated tumour necrosis factor alpha (TNF-α)- or phorbol myristate acetate (PMA)-induced activity of a nuclear factor κB (NF-κB)-luciferase reporter but did not interfere with the activity of an activator protein 1 (AP-1)-luciferase reporter. Phosphorylation of IκBα and nuclear translocation of NF-κB were also impaired by ISKNV ORF124L. In summary, ORF124L was identified as a vANK repeat protein and its role in inhibition of TNF-α-induced NF-κB signalling was investigated through interaction with the mandarin fish IKKß. This work may help to improve our understanding of the function of fish iridovirus ANK repeat proteins.

Proteomic analysis of Chilo iridescent virus.

In this first proteomic analysis of an invertebrate iridovirus, 46 viral proteins were detected in the virions of Chilo iridescent virus (CIV) based on the detection of 2 or more distinct peptides; an additional 8 proteins were found based on a single peptide. Thirty-six of the 54 identified proteins have homologs in another invertebrate and/or in one or more vertebrate iridoviruses. The genes for 5 of the identified proteins, 22L (putative helicase), 118L, 142R (putative RNaseIII), 274L (major capsid protein) and 295L, are shared by all iridoviruses for which the complete nucleotide sequence is known and may therefore be considered as iridovirus core genes. Three identified proteins have homologs only in ascoviruses. The remaining 15 identified proteins are so far unique to CIV. In addition to broadening our insight in the structure and assembly of CIV virions, this knowledge is pivotal to unravel the initial steps in the infection process.

Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV).

Infectious spleen and kidney necrosis virus (ISKNV) is a typical species of the genus Megalocytivirus in the family Iridoviridae. However, until recently, no suitable piscine cell line is stably susceptible to ISKNV. Here, a continuous cell culture derived from the mandarin fish fry (MFF-1) was developed and has been subcultured over 60 passages during a period of 18 months. MFF-1 consists predominantly of epithelial-like cells and grows well in DMEM supplemented with 10% fetal bovine serum. MFF-1 could produce high titers of ISKNV by continuous viral passages which were further confirmed by indirect immunofluorescence assay and RT-PCR analysis. Flow cytometry analyse showed that approximately 80.3% cells could be infected by ISKNV at 3 days post-infection. Abundant ISKNV particles were observed in the cytoplasm of the ISKNV infected MFF-1 cells by transmission electron microscopy. Mandarin fish injected with the filtrate from the infected cell suspension developed clinical signs and died, which is in accordance with the infectious spleen and kidney necrosis virus disease (ISKNVD). In addition, apoptosis was observed in the MFF-1 cells upon ISKNV infection by FITC-annexin V staining. ISKNV was purified and the viral protein profiles were also determined in this research. To our knowledge, MFF-1 is the first cell line originated from mandarin fish and it can be an efficient tool for the study of ISKNV.

Characterization of a membrane protein (VP001L) from infectious spleen and kidney necrosis virus (ISKNV).

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of megalocytivirus, Iridoviridae. A novel membrane protein corresponding to the first open reading frame (ORF001L) of ISKNV genome was identified. This 378-residue protein, termed the VP001L protein, has a high content of hydrophobic sequences and contains 10-11 putative transmembrane domains, indicating it may be a membrane protein. The VP001L mRNA start site was extended 433 bp upstream of the start codon and the temporal analysis showed that the VP001L gene was first transcribed at 8 h post-infection (h.p.i.). VP001L protein was detected on the plasma membrane of ISKNV infected cells by immunofluresence. In order to further investigate different transmembrane domains' influence on subcellular localization of VP001L, series of truncated or deleted mutants were constructed with GFP at the C terminus. The transfection results indicated that the second putative transmembrane domain played a determinative role in VP001L's membrane localization and the translocation of the first and third transmembrane domains depended on their interactions with the second one. Therefore, this novel VP001L protein is considered to serve as a model for analyzing the topology and roles of different hydrophobic regions in multi-transmembrane proteins.

Transmission of viruses to mosquito larvae mediated by divalent cations.

The two major groups of pathogenic viruses in mosquitoes are the occluded viruses, represented by baculoviruses and cypoviruses, and the non-occluded viruses, represented by the densoviruses and the iridoviruses. Baculoviruses, densoviruses, and iridoviruses are DNA viruses, while cypoviruses are the major group of RNA viruses reported from mosquitoes. Research on mosquito pathogenic viruses has been limited, in part, due to the inability to effectively transmit them to the larval mosquito host. Recently, there have been tremendous advancements in the ability to transmit mosquito baculoviruses and cypoviruses with the finding that transmission is mediated by divalent cations. Oral transmission of both baculoviruses and cypoviruses to mosquito larvae is enhanced by magnesium and inhibited by calcium ions. The current status of transmission for each of the major groups is reviewed with emphasis on the common role of divalent cations in transmission of the distantly related baculoviruses and cypoviruses.

Transcriptional mapping in Chilo iridescent virus infections.

Chilo iridescent virus (CIV) belongs to the family Iridoviridae, which are icosahedral cytoplasmic DNA viruses with large, linear, and circularly permuted genomes. Previous studies on infected-cell-specific polypeptides suggested temporal regulation of CIV gene expression. Recently, we demonstrated three temporal classes at the transcriptional level, in CIV infections of a spruce budworm cell line. We also demonstrated a transcriptional cascade with positive and negative control. In this paper, we assign all detectable viral transcripts into respective temporal classes and map them using restriction fragments from a genomic library. More than 90 percent of the genome is transcriptionally active with at least four major clusters of immediate-early transcription and at least three delayed-early clusters. Late transcripts were observed throughout the genome. There was at least one exclusive region in the genome for each of the three temporal classes. We correlated transcribed regions with ORFs on the CIV genome and showed that known ORFs in the exclusive regions are generally consistent with phase-specific requirements of large DNA viruses. Our data also suggest the presence of 5' or 3' coterminal transcripts. This is the first complete transcription map for a member of the genus Iridovirus.

The familiar and the unexpected in structures of icosahedral viruses.

Viruses were the first large macromolecular assemblages to be visualized at high resolution. New virus structures continue to challenge our understanding of specificity in protein-protein "recognition". The evolution of virus structures has been even more opportunistic than previously imagined.