Near-atomic architecture of Singapore grouper iridovirus and implications for giant virus assembly.

Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide. Effective control and prevention strategies are urgently needed. Here, we present a near-atomic architecture of the SGIV capsid and identify eight types of capsid proteins. The viral inner membrane-integrated anchor protein colocalizes with the endoplasmic reticulum (ER), supporting the hypothesis that the biogenesis of the inner membrane is associated with the ER. Additionally, immunofluorescence assays indicate minor capsid proteins (mCPs) could form various building blocks with major capsid proteins (MCPs) before the formation of a viral factory (VF). These results expand our understanding of the capsid assembly of NCVs and provide more targets for vaccine and drug design to fight iridovirid infections.

Singapore Grouper Iridovirus VP131 Drives Degradation of STING-TBK1 Pathway Proteins and Negatively Regulates Antiviral Innate Immunity.

Iridoviruses are large DNA viruses which cause great economic losses to the aquaculture industry and serious threats to ecological diversity worldwide. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, causes high mortality in grouper aquaculture. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. Here, we reported that the protein encoded by SGIV ORF131R (VP131) was localized predominantly within the endoplasmic reticulum (ER). Ectopic expression of GFP-VP131 significantly enhanced SGIV replication, while VP131 knockdown decreased viral infection in vitro, suggesting that VP131 functioned as a proviral factor during SGIV infection. Overexpression of GFP-VP131 inhibited the interferon (IFN)-1 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), TANK-binding kinase 1 (EcTBK1), or melanoma differentiation-associated gene 5 (EcMDA5), whereas such activation induced by mitochondrial antiviral signaling protein (EcMAVS) was not affected. Moreover, VP131 interacted with EcSTING and degraded EcSTING through both the autophagy-lysosome pathway and ubiquitin-proteasome pathway, and targeted for the K63-linked ubiquitination. Of note, we also found that EcSTING significantly accelerated the formation of GFP-VP131 aggregates in co-transfected cells. Finally, GFP-VP131 inhibited EcSTING- or EcTBK1-induced antiviral activity upon red-spotted grouper nervous necrosis virus (RGNNV) infection. Together, our results demonstrated that the SGIV VP131 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion. IMPORTANCE STING has been identified as a critical factor participating in the innate immune response which recruits and phosphorylates TBK1 and IFN regulatory factor 3 (IRF3) to induce IFN production and defend against viral infection. However, viruses also distort the STING-TBK1 pathway to negatively regulate the IFN response and facilitate viral replication. Here, we reported that SGIV VP131 interacted with EcSTING within the ER and degraded EcSTING, leading to the suppression of IFN production and the promotion of SGIV infection. These results for the first time demonstrated that fish iridovirus evaded the host antiviral response via abrogating the STING-TBK1 signaling pathway.

Glycosylphosphatidylinositol Mannosyltransferase â Protects Chinese Giant Salamander, Andrias davidianus, against Iridovirus.

Glycosylphosphatidylinositol mannosyltransferase I (GPI-MT-I) is an essential glycosyltransferase of glycosylphosphatidylinositol-anchor proteins (GPI-APs) that transfers the first of the four mannoses in GPI-AP precursors, which have multiple functions, including immune response and signal transduction. In this study, the GPI-MT-I gene that regulates GPI-AP biosynthesis in Andrias davidianus (AdGPI-MT-I) was characterized for the first time. The open reading frame (ORF) of AdGPI-MT-I is 1293 bp and encodes a protein of 430 amino acids that contains a conserved PMT2 superfamily domain. AdGPI-MT-I mRNA was widely expressed in the tissues of the Chinese giant salamander. The mRNA expression level of AdGPI-MT-I in the spleen, kidney, and muscle cell line (GSM cells) was significantly upregulated post Chinese giant salamander iridovirus (GSIV) infection. The mRNA expression of the virus major capsid protein (MCP) in AdGPI-MT-I-overexpressed cells was significantly reduced. Moreover, a lower level of virus MCP synthesis and gene copying in AdGPI-MT-I-overexpressed cells was confirmed by western blot and ddPCR. These results collectively suggest that GSIV replication in GSM cells was significantly reduced by the overexpression of the AdGPI-MT-I protein, which may contribute to a better understanding of the antiviral mechanism against iridovirus infection.

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.

The Roles of Epinephelus coioides miR-122 in SGIV Infection and Replication.

In mammals, mature miR-122 is 22 nucleotides long and can be involved in regulating a variety of physiological and biological pathways. In this study, the expression profile and effects of grouper Epinephelus coioides miR-122 response to Singapore grouper iridovirus (SGIV) infection were investigated. The sequences of mature microRNAs (miRNAs) from different organisms are highly conserved, and miR-122 from E. coioides exhibits high similarity to that from mammals and other fish. The expression of miR-122 was up-regulated during SGIV infection. Up-regulation of miR-122 could significantly enhance the cytopathic effects (CPE) induced by SGIV, the transcription levels of viral genes (MCP, VP19, LITAF and ICP18), and viral replication; reduce the expression of inflammatory factors (TNF-a, IL-6, and IL-8), and the activity of AP-1 and NF-κB, and miR-122 can bind the target gene p38α MAPK to regulate the SGIV-induced cell apoptosis and the protease activity of caspase-3. The results indicated that SGIV infection can up-regulate the expression of E. coioides miR-122, and up-regulation of miR-122 can affect the activation of inflammatory factors, the activity of AP-1 and NF-κB, and cell apoptosis to regulate viral replication and proliferation.

The interactome of Singapore grouper iridovirus protein ICP18 as revealed by proximity-dependent BioID approach.

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that is a major threat to grouper aquaculture. The pathogenesis of SGIV is not well understood so far. Previous studies have revealed that ICP18, an immediate early protein encoded by SGIV ORF086R gene, promotes viral replication by regulating cell proliferation and virus assembly. In the present study, the potential functions of ICP18 were further explored by probing into its interactors using a proximity-dependent BioID method. Since our in-house grouper embryonic cells (a natural host cell of SGIV) could not be efficiently transfected with the plasmid DNA, and the grouper genome data for mass spectrometry-based protein identification is not currently available, we chosen a non-permissive cell (HEK293 T) as a substitute for this study. A total of 112 cellular proteins that potentially bind to ICP18 were identified by mass spectrometry analysis. Homology analysis showed that among these identified proteins, 110 candidate ICP18-interactors had homologous proteins in zebrafish (a host of SGIV), and shared high sequence identity. Further analysis revealed that the identified ICP18-interacting proteins modulate various cellular processes such as cell cycle and cell adhesion. In addition, the interaction between ICP18 and its candidate interactor, i.e., cyclin-dependent kinase1 (CDK1), was confirmed using Co-immunoprecipitation (Co-IP) and Pull-down assays. Collectively, our present data provides additional insight into the biological functions of ICP18 during viral infection, which could help in further unraveling the pathogenesis of SGIV.

Chinese Giant Salamander (Andrias davidianus) Iridovirus Infection Leads to Apoptotic Cell Death through Mitochondrial Damage, Caspases Activation, and Expression of Apoptotic-Related Genes.

Chinese giant salamander iridovirus (GSIV) is the causative pathogen of Chinese giant salamander (Andrias davidianus) iridovirosis, leading to severe infectious disease and huge economic losses. However, the infection mechanism by GSIV is far from clear. In this study, a Chinese giant salamander muscle (GSM) cell line is used to investigate the mechanism of cell death during GSIV infection. Microscopy observation and DNA ladder analysis revealed that DNA fragmentation happens during GSIV infection. Flow cytometry analysis showed that apoptotic cells in GSIV-infected cells were significantly higher than that in control cells. Caspase 8, 9, and 3 were activated in GSIV-infected cells compared with the uninfected cells. Consistently, mitochondria membrane potential (MMP) was significantly reduced, and cytochrome c was released into cytosol during GSIV infection. p53 expression increased at an early stage of GSIV infection and then slightly decreased late in infection. Furthermore, mRNA expression levels of pro-apoptotic genes participating in the extrinsic and intrinsic pathway were significantly up-regulated during GSIV infection, while those of anti-apoptotic genes were restrained in early infection and then rose in late infection. These results collectively indicate that GSIV induces GSM apoptotic cell death involving mitochondrial damage, caspases activation, p53 expression, and pro-apoptotic molecules up-regulation.

Segmentation and Comparative Modeling in an 8.6-Å Cryo-EM Map of the Singapore Grouper Iridovirus.

SGIV, or Singapore grouper iridovirus, is a large double-stranded DNA virus, reaching a diameter of 220 nm and packaging a genome of 140 kb. We present a 3D cryoelectron microscopy (cryo-EM) icosahedral reconstruction of SGIV determined at 8.6-Å resolution. It reveals several layers including a T = 247 icosahedral outer coat, anchor proteins, a lipid bilayer, and the encapsidated DNA. A new segmentation tool, iSeg, was applied to extract these layers from the reconstructed map. The outer coat was further segmented into major and minor capsid proteins. None of the proteins extracted by segmentation have known atomic structures. We generated models for the major coat protein using three comparative modeling tools, and evaluated each model using the cryo-EM map. Our analysis reveals a new architecture in the Iridoviridae family of viruses. It shares similarities with others in the same family, e.g., Chilo iridescent virus, but also shows new features of the major and minor capsid proteins.

Identification and initial functional characterization of lysosomal integral membrane protein type 2 (LIMP-2) in turbot (Scophthalmus maximus L.).

The immune system protects organism from external pathogens, this progress starts with the pathogen recognition by pattern recognition receptors (PRRs). As a group of PRRs, the class B scavenger receptors showed important roles in phagocytosis. Among three class B scavenger receptors, lysosomal integral membrane protein type 2 (LIMP-2) was reported to present in the limiting membranes of lysosomes and late endosomes, but its immune roles in teleost species are still limited in handful species. Here, we characterized LIMP-2 gene in turbot, and its expression patterns in mucosal barriers following different bacterial infection, as well as ligand binding activities to different microbial ligands and agglutination assay with different bacteria. In our results, one SmLIMP2 gene was identified with a 1,593 bp open reading frame (ORF). The multiple species comparison and phylogenetic analysis showed the closest relationship to Paralichthys olivaceus, the genomic structure analysis and syntenic analysis revealed the conservation of LIMP-2 during evolution. In tissue distribution analysis, SmLIMP-2 was expressed in all the examined turbot tissues, with the highest expression level in brain, and the lowest expression level in liver. In addition, SmLIMP-2 was significantly up-regulated in all the mucosal tissues (skin, gill and intestine) following Gram-negative bacteria Vibrio anguillarum infection, and was only up-regulated in gill following Gram-positive bacteria Streptococcus iniae challenge. Finally, the rSmLIMP-2 showed strong binding ability to all the examined microbial ligands, and strong agglutination with Escherichia coli, Staphylococcus aureus and V. anguillarum. Taken together, our results suggested SmLIMP-2 played important roles in fish immune response to bacterial infection. However, further functional studies should be carried out to better characterize its detailed roles in teleost immunity.

Grouper iridovirus GIV66 is a Bcl-2 protein that inhibits apoptosis by exclusively sequestering Bim.

Programmed cell death or apoptosis is a critical mechanism for the controlled removal of damaged or infected cells, and proteins of the Bcl-2 family are important arbiters of this process. Viruses have been shown to encode functional and structural homologs of Bcl-2 to counter premature host-cell apoptosis and ensure viral proliferation or survival. Grouper iridovirus (GIV) is a large DNA virus belonging to the Iridoviridae family and harbors GIV66, a putative Bcl-2-like protein and mitochondrially localized apoptosis inhibitor. However, the molecular and structural basis of GIV66-mediated apoptosis inhibition is currently not understood. To gain insight into GIV66's mechanism of action, we systematically evaluated its ability to bind peptides spanning the BH3 domain of pro-apoptotic Bcl-2 family members. Our results revealed that GIV66 harbors an unusually high level of specificity for pro-apoptotic Bcl-2 and displays affinity only for Bcl-2-like 11 (Bcl2L11 or Bim). Using crystal structures of both apo-GIV66 and GIV66 bound to the BH3 domain from Bim, we unexpectedly found that GIV66 forms dimers via an interface that results in occluded access to the canonical Bcl-2 ligand-binding groove, which breaks apart upon Bim binding. This observation suggests that GIV66 dimerization may affect GIV66's ability to bind host pro-death Bcl-2 proteins and enables highly targeted virus-directed suppression of host apoptosis signaling. Our findings provide a mechanistic understanding for the potent anti-apoptotic activity of GIV66 by identifying it as the first single-specificity, pro-survival Bcl-2 protein and identifying a pivotal role of Bim in GIV-mediated inhibition of apoptosis.

Singapore grouper iridovirus protein VP088 is essential for viral infectivity.

Viral infection is a great challenge in healthcare and agriculture. The Singapore grouper iridovirus (SGIV) is highly infectious to numerous marine fishes and increasingly threatens mariculture and wildlife conservation. SGIV intervention is not available because little is known about key players and their precise roles in SGVI infection. Here we report the precise role of VP088 as a key player in SGIV infection. VP088 was verified as an envelope protein encoded by late gene orf088. We show that SGIV could be neutralized with an antibody against VP088. Depletion or deletion of VP088 significantly suppresses SGIV infection without altering viral gene expression and host responses. By precisely quantifying the genome copy numbers of host cells and virions, we reveal that VP088 deletion dramatically reduces SGIV infectivity through inhibiting virus entry without altering viral pathogenicity, genome stability and replication and progeny virus release. These results pinpoint that VP088 is a key player in SGIV entry and represents an ideal target for SGIV intervention.

GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these results suggested that aquatic GSIV ST kinase could induce apoptosis via upregulation of p53 and Bax expression, resulting in mitochondrial disruption, which activated a downstream caspases-mediated cell death pathway.

Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways.

Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways.

Monoclonal antibody against a putative myristoylated membrane protein encoded by grouper iridovirus 59L gene.

Groupers (Epinephelus spp.) are economically important fish species worldwide, and ranaviruses are major viral pathogens causing heavy economic losses in grouper aquaculture. In this study, the 59L gene of grouper iridovirus (GIV-59L) was cloned and characterized. This gene is 1521 bp and encodes a protein of 506 amino acids with a predicted molecular mass of 53.9 kDa. Interestingly, GIV-59L and its homologs are found in all genera of the family Iridoviridae. A mouse monoclonal antibody specific for the C-terminal domain (amino acid positions 254-506) of the GIV-59L protein, GIV-59L(760-1518)-MAb-21, was produced and proved to be well suited for use in a number of GIV immunoassays. RT-PCR, Western blotting, and cycloheximide and cytosine arabinoside drug inhibition analyses indicated that GIV-59L is a viral late gene in GIV-infected grouper kidney cells. Immunofluorescence analysis revealed that GIV-59L protein mainly accumulates in the cytoplasm of infected cells and is finally packed into a whole virus particle. The GIV-59L(760-1518)-MAb-21 characterized in this study could have widespread application in GIV immunodiagnostics and other research on GIV. In addition, the results presented here offer important insights into the pathogenesis of GIV.

Cloning, sequence analysis and expression profiles of Toll-like receptor 7 from Chinese giant salamander Andrias davidianus.

The Chinese giant salamander, Andrias davidianus, is the largest extant amphibian species in the world, which is of significance due to its specific position in the evolutionary history of vertebrates. Currently, limited information about the innate immune system of this animal is known. In this study, the toll-like receptor 7 (TLR7), designated CgsTLR7, was cloned from Chinese giant salamander, A. davidianus. The full-length cDNA of CgsTLR7 is 3747 bp, with an open reading frame of 3150 bp, encoding 1049 amino acids. The TLR family motifs, including the leucine-rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain are conserved in CgsTLR7, which includes 19 LRRs and a TIR domain. The predicted amino acid sequence of CgsTLR7 has 71%, 65%, 63% and 55% identity with turtle, chicken, human and fugu TLR7 homologues, respectively. Phylogenetic analysis showed that CgsTLR7 is closest to that of frog TLR7 among the examined species. Quantitative real-time PCR analysis revealed broad expression of CgsTLR7 in tissues from apparently healthy Chinese giant salamanders with the highest expression in the liver and the lowest expression in the intestine. The mRNA expression was up-regulated and reached a peak level in the kidney, liver and spleen at 12 h, 24 h and 48 h after infecting the animals with the giant salamander iridovirus (GSIV), respectively. These results suggest that CgsTLR7 has a conserved gene structure and might play an important role in immune regulation against viral infections in the Chinese giant salamander.

Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

Temporal proteomic analysis and label-free quantification of viral proteins of an invertebrate iridovirus.

Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a ~212 kb linear dsDNA genome that encodes 215 putative ORFs. The IIV-6 virion-associated proteins consist of at least 54 virally encoded proteins. One of our previous findings showed that most of these proteins are encoded by genes from the early transcriptional class. This indicated that these structural proteins may not only function in the formation of the virion, but also in the initial stage of viral infection. In the current study, we followed the protein expression profile of IIV-6 over time in Drosophila S2 cells by label-free quantification using a proteomic approach. A total of 95 virally encoded proteins were detected in infected cells, of which 37 were virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles: proteins with stably low expression levels during infection, proteins with exponentially increasing expression levels during infection and proteins that were initially highly abundant, but showed slightly reduced levels after 48 h post-infection. We thus provided novel information on the kinetics of virion and infected cell-specific protein levels that assists in our understanding of gene regulation in this lesser-known DNA virus model.

Development and characterization of two monoclonal antibodies against grouper iridovirus 55L and 97L proteins.

Grouper iridovirus (GIV) is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. The study of GIV pathogenicity has been hampered by the lack of proper immunological reagents to study the expression and function of viral proteins in the infected cells. In this study, two mouse monoclonal antibodies (mAbs) against GIV 55L and 97L proteins were produced. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen these hybridomas, resulting in the identification of two high-affinity mAbs named GIV55L-mAb-2 and GIV97L-mAb-3, respectively. Both mAbs belong to the IgG1 isotype and were effective in detecting their respective target viral protein. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analyses of GIV-infected GK cells revealed that GIV 97L is an immediate early gene, whereas GIV 55L a late one. The localization of 55L and 97L in GIV-infected cells was further characterized by immunofluorescence microscopy with the mAbs. The 55L protein mainly aggregated in the cytoplasm while 97L distributed in both the nucleus and cytoplasm of the infected cells. These studies demonstrate the validity of the two mAbs as immunodiagnostic and research reagents.

Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus).

Prokaryotes contain many DNA binding proteins with large molecular weights and multiple domains. DNA binding proteins are involved in DNA replication, transcription, and other physiological processes. In this study, a DNA binding protein, containing an Ftsk-like protein (FLP) domain, was cloned and characterized from SKIV-ZJ07, a member of the RSIV-type megalocytivirus, using bioinformatics and molecular biology approaches. SKIV-FLP is 3,762 base pairs long, encodes a viral protein of 1253 amino acid residuals, and contains an Ftsk (or EBV-NA3) and a Grx-2 domain. Virion localization indicated that SKIV-FLP is a major viral structural protein located below the major capsid protein. Laser confocal microscopy showed that SKIV-FLP is a cytoplasm-/nuclear-localized protein. However, the reconstruction experiments demonstrated that SKIV-FLP may contain three nuclear localization signals, each present in FLP-NT (1-380 aa), FtsK domain (380-880 aa), and Grx-2 domain (880-1253 aa). When SKIV-FLP was fused to the Gal-4 DNA-binding domain and co-transfected with L8G5-Luc, SKIV-FLP suppressed L8G5-Luc transcription. As a transcription inhibitor, SKIV-FLP also inhibited the transcription of NF-κB and IFN-γ (a type II IFN) promoter in HEK293T cells, suggesting that SKIV-FLP has a role in evading host immunity.

An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication.

Insulin-like growth factors (IGFs) play crucial roles in regulating cell differentiation, proliferation and apoptosis. In this study, a novel IGF homologue gene (IGF-like) encoded by Singapore grouper iridovirus (SGIV) ORF062R (termed SGIV-IGF), was cloned and characterized. The coding region of SGIV-IGF is 771 bp in length, with a variable number of tandem repeats (VNTR) locus at the 3'-end. We cloned one isoform of this novel gene, 582 bp in length, containing the predicted IGF domain and 3.6 copy numbers of the 27 bp repeat unit. SGIV-IGF was an early transcribed gene during viral infection, and SGIV-IGF was distributed predominantly in the cytoplasm with a diffused granular appearance. Intriguingly, overexpression of SGIV-IGF was able to promote the growth of grouper embryonic cells (GP cells) by promoting G1/S phase transition, which was at least partially dependent on its 3'-end VNTR locus. Furthermore, viral titre assay and real-time quantitative PCR (RT-qPCR) analysis proved that SGIV-IGF could promote SGIV replication in grouper cells. In addition, overexpression of SGIV-IGF mildly facilitated apoptosis in SGIV-infected non-host fathead minnow (FHM) cells. Together, our study demonstrated a novel functional gene of SGIV which may regulate viral replication and cellular processes through multiple mechanisms that appear to be cell type-dependent.