Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus.

We recently reported that grouper iridovirus (GIV) can induce apoptosis in barramundi, Lates calcarifer, muscle (BM) and swim bladder (BSB) cell lines. In this paper, we further characterize the molecular mechanism underlying apoptotic death in BM cells triggered by GIV. DNA-laddering and apoptotic cells were observed in BM cells infected with UV-irradiated or untreated GIV but was absent in cells infected with heat-inactivated GIV, indicating the involvement of viral protein in the apoptosis event. In GIV-infected BM cells, the conversion of procaspase-3 to caspase-3 was evident and the level of caspase-8 and -9 increased as early as 30 min post-infection. When treated with a pancaspase inhibitor, the GIV-induced apoptosis event was abolished. These observations indicate that GIV-induced apoptosis is caspase-dependent, and that both the external and internal routes in the caspase-dependent pathway are likely involved in the apoptosis process.

RING finger proteins of infectious spleen and kidney necrosis virus (ISKNV) function as ubiquitin ligase enzymes.

Infectious spleen and kidney necrosis virus (ISKNV) is the etiological agent that causes a pandemic and severe disease in fish characteristic of enlarged and damaged spleen and kidney. To identify viral proteins involved in infection and pathogenesis, we characterized five open reading frames (ORFs) of the ISKNV genome, ORF12, ORF65, ORF66, ORF99 and ORF111, which encode RING finger proteins (RFPs). We assessed the ubiquitin ligase (E3) activity of these recombinant RFPs fused to maltose-binding protein (MBP) using an in vitro ubiquitination assay and demonstrated that ORF12, ORF65, ORF66 and ORF111 possess the E3 activity in the presence of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin and zinc ion. E3 activity of ISKNV RFPs strictly depends on the UbcH5 E2 subfamily (ORF12 and ORF65 depend on UbcH5a/c, ORF66 and ORF111 depend on UbcH5a/b/c). Furthermore, point mutation in the RING domain completely abrogated ORF66 E3 activity, indicating the RING motif was essential for RFP of ISKNV. In addition, zinc ion was required as an enhancer for ISKNV RFP to exert its E3 function. Investigation of RFPs of ISKNV helps to understand their functions in the infection process and in the virus-host interaction.

Identification, mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus.

The thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) was identified by biochemical transformation of 3T3 TK negative (TK-) to 3T3 TK positive (TK+) cells using specific viral DNA sequences. DNA fragments of the viral genome used in this study were obtained from a defined gene library of FLDV genome containing the complete viral DNA sequences. The selection of the converted cells was carried out under the condition of the HAT selection procedure. The results of these experiments revealed that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611 to 0.718 map units) is able to transform 3T3 TK- to 3T3 TK+ cells. Additional experiments using the subclones of EcoRI DNA fragment C revealed that DNA sequences of 4.1 kbp size between the coordinates 0.669 to 0.718 of the FLDV genome possessed the ability for biochemical transformation, indicating that the TK gene locus is located in this particular region.

[Virion-associated enzymes of the iridovirus in the mosquito Aedes cantans]. / Virionassotsiirovannye fermenty iridovirusa komara Aedes cantans.

Purified virions of iridovirus of Aedes cantans mosquito have proteinkinase and ATP-ase activities. Protein kinase associated with highly purified virions of this virus is cAMP-dependent, ATP-ase of mosquito iridovirus is Mg2+-dependent. Its activity is not affected by Na+ and K+ ions, whereas Ca2+ ions inhibit it.

Evolution of DNA ligases of nucleo-cytoplasmic large DNA viruses of eukaryotes: a case of hidden complexity.

BACKGROUND: Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) encode most if not all of the enzymes involved in their DNA replication. It has been inferred that genes for these enzymes were already present in the last common ancestor of the NCLDV. However, the details of the evolution of these genes that bear on the complexity of the putative ancestral NCLDV and on the evolutionary relationships between viruses and their hosts are not well understood. RESULTS: Phylogenetic analysis of the ATP-dependent and NAD-dependent DNA ligases encoded by the NCLDV reveals an unexpectedly complex evolutionary history. The NAD-dependent ligases are encoded only by a minority of NCLDV (including mimiviruses, some iridoviruses and entomopoxviruses) but phylogenetic analysis clearly indicated that all viral NAD-dependent ligases are monophyletic. Combined with the topology of the NCLDV tree derived by consensus of trees for universally conserved genes suggests that this enzyme was represented in the ancestral NCLDV. Phylogenetic analysis of ATP-dependent ligases that are encoded by chordopoxviruses, most of the phycodnaviruses and Marseillevirus failed to demonstrate monophyly and instead revealed an unexpectedly complex evolutionary trajectory. The ligases of the majority of phycodnaviruses and Marseillevirus seem to have evolved from bacteriophage or bacterial homologs; the ligase of one phycodnavirus, Emiliana huxlei virus, belongs to the eukaryotic DNA ligase I branch; and ligases of chordopoxviruses unequivocally cluster with eukaryotic DNA ligase III. CONCLUSIONS: Examination of phyletic patterns and phylogenetic analysis of DNA ligases of the NCLDV suggest that the common ancestor of the extant NCLDV encoded an NAD-dependent ligase that most likely was acquired from a bacteriophage at the early stages of evolution of eukaryotes. By contrast, ATP-dependent ligases from different prokaryotic and eukaryotic sources displaced the ancestral NAD-dependent ligase at different stages of subsequent evolution. These findings emphasize complex routes of viral evolution that become apparent through detailed phylogenomic analysis but not necessarily in reconstructions based on phyletic patterns of genes. REVIEWERS: This article was reviewed by: Patrick Forterre, George V. Shpakovski, and Igor B. Zhulin.

Constitutive expression of thymidylate synthase from LCDV-C induces a transformed phenotype in fish cells.

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumor formation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virus from China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4-5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity.

Characterization of the Rana grylio virus 3beta-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect.

The 3beta-hydroxysteroid dehydrogenase (3beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Here, a 3beta-HSD gene homolog was cloned from Rana grylio virus (RGV), a member of family Iridoviridae. RGV 3beta-HSD gene has 1068bp, encoding a 355aa predicted protein. Transcription analyses showed that RGV 3beta-HSD gene was transcribed immediate-early during infection from an initiation site 19 nucleotides upstream of the translation start site. Confocal microscopy revealed that the 3beta-HSD-EGFP fusion protein was exclusively colocalized with the mitochondria marker (pDsRed2-Mito) in EPC cells. Upon morphological observation and MTT assay, it was revealed that overexpression of RGV 3beta-HSD in EPC cells could apparently suppress RGV-induced cytopathic effect (CPE). The present studies indicate that the RGV immediate-early 3beta-HSD gene encodes a mitochondria-localized protein, which has a novel role in suppressing virus-induced CPE. All these suggest that RGV 3beta-HSD might be a protein involved in host-virus interaction.

Evidence for an acid phosphatase in African swine fever virus.

An acid phosphatase activity has been detected in purified preparations of African swine fever virus. Purified viral cores obtained after Nonidet-P40 and 2-mercaptoethanol treatment of the virus retained the activity as assayed with nitrophenyl phosphate as substrate at pH 5. Enzyme cytochemistry by electron microscopy showed that the acid phosphatase activity is localized mainly inside the core of the virion. The molecular weight and the isoelectric point of the virus acid phosphatase activity confirmed that it was distinct from the host cellular enzyme.

Identification of the gene encoding the DNA (cytosine-5) methyltransferase of lymphocystis disease virus.

The gene encoding the DNA (cytosine-5) methyltransferase (m5C-MTase) of lymphocystis disease virus (flounder isolate, LCDV-1) has been identified by polymerase chain reaction (PCR) using oligonucleotide primers synthesized corresponding to different regions of the m5C-MTase gene of frog virus 3 (FV3). A DNA fragment of 487 bp was amplified using oligonucleotide primers L3 and R4 which correspond to the nucleotide positions 87 to 109 and 530 to 550 of the m5C-MTase gene of FV3, respectively. The DNA nucleotide sequence of the PCR product was determined by direct cycle sequencing. The alignment of the deduced amino acid sequence derived from the PCR product and the m5C-MTase protein of FV3 revealed a homology of 55.4% identity and 29.1% similarity. The amino acid sequence which was found to be significantly homologous to the amino acid sequence deduced from the nucleotide sequence of the PCR product was located at the amino acid position 37 to 175 of the m5C-MTase of FV3 indicating the specificity of the amplified PCR product. The DNA nucleotide sequence of the LCDV-1 genome corresponding to the 5' and 3' termini of the m5C-MTase gene was determined by primer walking. The locus of the m5C-MTase gene of LCDV-1 was identified within the EcoRI DNA fragment G of LCDV-1 (7.9 kbp; 0.947 to 0.034 map units). The m5C-MTase gene of LCDV-1 comprises 684 nucleotides coding for a putative protein of 228 amino acid residues. A high degree of amino acid sequence homology (53.3% identity and 25.8% similarity) was detected between the m5C-MTase of LCDV-1 and FV3.

DNA methyltransferase induced by frog virus 3.

Over 20% of the cytosine bases in frog virus 3 DNA are methylated at the 5-carbon position. To determine whether this high degree of methylation is the result of a virus-specific enzyme, we examined the kinetics of induction and the substrate specificity of a DNA methyltransferase from frog virus 3-infected fathead minnow cells. A novel DNA methyltransferase activity appeared in the cytoplasm of infected cells at 3 h postinfection. This activity was induced in the absence of viral DNA replication and was therefore probably an early viral enzyme. In contrast to the methyltransferase activity extracted from uninfected cell nuclei, the cytoplasmic enzyme showed a strong template preference for double-stranded over single-stranded and for unmethylated over hemimethylated DNA. The dinucleotide sequence dCpdG was a necessary and sufficient exogenous substrate for methylation in vitro. A mutant of frog virus 3, isolated as resistant to 5-azacytidine and having unmethylated virion DNA, did not induce cytoplasmic DNA methyltransferase, leading to the conclusion that this activity is coded for by the virus.

Metabolism of host and viral mRNAs in frog virus 3-infected cells.

Treatment of purified frog virus 3 (FV3) with nonionic detergent and high salt released an endoribonucleolytic activity and confirmed earlier findings of a virion-associated endonuclease. This observation, coupled with evidence implicating host and viral message destabilization in herpesvirus and poxvirus biogenesis, raised the question of what role, if any, mRNA degradation plays in FV3 replication. To answer this question, Northern analyses of mock- and virus-infected cells were performed using probes for representative host and viral messages. These studies demonstrated that the steady state level of host messages progressively declined during the course of productive FV3 infection, whereas the steady state level of viral messages was not affected. To determine whether the decline in the steady state level of host mRNA was due to virus-induced degradation or to normal turnover coupled to virus-mediated transcriptional shut-off, actin mRNA levels were examined in mock- and virus-infected cells in the presence and absence of actinomycin D. Under these conditions, actin mRNA levels declined more quickly in actinomycin D-treated, virus-infected cells, than in mock-infected cells incubated in the presence of actinomycin D suggesting that the decline in the steady state level of actin mRNA was due to degradation. However, although it appears as if host message degradation is responsible for virus-mediated translational shut-off, the ability of heat-inactivated FV3 to block cellular translation without destabilizing cellular messages indicates that message degradation is not required for translational inhibition. As noted above, the degradation of early FV3 messages was not involved in controlling the transition from early to late gene expression. Furthermore, the presence of abundant, but nontranslated, early messages late in infection, coupled with the inefficient translation of late messages in vitro supported earlier suggestions that FV3 gene expression is controlled, at least in part, at the translational level. Taken together, these results suggest that FV3 regulates gene expression in a unique manner and may be a good model to examine the mechanics of translational control.

Further characterization of an alkaline protease activity associated with iridescent virus type 6. Brief report.

Iridescent virus type 6 infecting Galleria mellonella larvae contained an associated alkaline protease activity which appeared to be tightly bound to the virions and essentially localized on the outside of the viral particle. Under alkaline conditions the virus was degraded by proteolytic cleavage of viral envelope proteins. Proteolytic activity was not present in virus propagated in a permissive insect cell line or when purified from isolated larval fat-bodies instead from whole larvae. The results suggest that the protease associated with Iridescent virus type 6 is of larval origin.

Single-stranded deoxyribonucleic acid nuclease induced by African swine fever virus and associated to the virion.

Infection of Vero cells with African swine fever (ASF) virus resulted in a marked increase of DNase active on single-stranded DNA (ss-DNase). No increase was observed for double-stranded DNA-specific nuclease activity. In contrast to uninfected cell ss-DNase, which has a pH optimum at pH range 8.5-9, virus-induced ss-DNase is most active at pH 7. Differences in sensitivity to several ions and other modifications of the reaction mixture and considerable difference in reaction kinetics suggest that the increase in nuclease activity is due to a new virus-induced enzyme. This is strengthened by the fact that anti-ASF virus antiserum inhibits the activity of ss-DNase from infected cells but not from uninfected cells. Exclusion chromatography of the digests shows that virus-specific ss-DNase is exclusively or predominantly an endonuclease. The increase in nuclease activity of infected cells is proportional to the multiplicity of infection. Virus-specific ss-DNase is synthesized at late times after infection and its synthesis is dependent on viral DNA replication since it is not induced when infected cells are treated with cytosine arabinoside. Most of ss-DNase activity in infected cells is associated to an insoluble cytoplasmic fraction, presumably virosomes. The enzyme can also be detected in partially stripped purified virions which hydrolyze 6.9 ng DNA per microgram viral protein.

Identification and properties of the largest subunit of the DNA-dependent RNA polymerase of fish lymphocystis disease virus: dramatic difference in the domain organization in the family Iridoviridae.

Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of the known largest subunits of DdRPs from different species contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains (RQP[T/S]LH and NADFDGDE) were used for detecting the corresponding gene of fish lymphocystis disease virus (FLCDV), a member of the family Iridoviridae, which replicates in the cytoplasm of infected cells of flatfish. The gene coding for the largest subunit of the DdRP was identified using a PCR-derived probe. The screening of the complete EcoRI gene library of the viral genome led to the identification of the gene locus of the largest subunit of the DdRP within the EcoRI DNA fragment B (12.4 kbp, 0.034 to 0.165 map units). The nucleotide sequence of a part (8334 bp) of the EcoRI DNA fragment B was determined and a large ORF on the lower strand (ATG = 5787; TAA = 2190) was detected which encodes a protein of 1199 amino acids. Comparison of the amino acid sequences of the largest subunits of the DdRP (RPO1) of FLCDV and Chilo iridescent virus (CIV) revealed a dramatic difference in their domain organization. Unlike the 1051 aa RPO1 of CIV, which lacks the C-terminal domain conserved in eukaryotic, eubacterial and other viral RNA polymerases, the 1199 aa RPO1 of FLCDV is fully collinear with its cellular and viral homologues. Despite this difference, comparative analysis of the amino acid sequences of viral and cellular RNA polymerases suggests a common origin for the largest RNA polymerase subunits of FLCDV and CIV.

Infection with frog virus 3 allows transcription of DNA methylated at cytosine but not adenine residues.

The genome of the iridovirus, frog virus 3, is highly methylated at cytosine residues by a virus-encoded DNA methyltransferase. We have shown previously that an FV3-induced trans-acting protein alters either host RNA polymerase II or methylated template to allow transcription from promoters inactivated by methylation. We now present evidence that the ability of FV3-infected cells to transcribe methylated DNA is specific for DNA methylated at cytosine residues. Eukaryotic promoters were inactivated by methylation of either adenine or cytosine residues, and tested for transcriptional activity. Only promoters inactivated by cytosine methylation were transcribed in FV3-infected cells. We also show that the dinucleotide sequence in which the methylcytosine is found appears to have no effect on the ability of FV3 to trans-activate the methylated promoters.

DNA sequences required for trans-activation of an immediate-early frog virus 3 gene.

A plasmid containing 78 bp of the promoter region of the immediate-early frog virus 3 (FV3) gene ICR 169 placed 5' to the coding sequences for chloramphenicol acetyltransferase (CAT) can only be induced to synthesize CAT after transfection in the presence of FV3. To determine what DNA sequences in the promoter were required for virus-induced transcription, I used site-directed mutagenesis to construct deletions and point mutations throughout the promoter region. The mutant promoters were then analyzed for their ability to be induced by FV3. Deletion of 27 bp from the 5' end of the promoter had little effect on FV3-induced CAT synthesis. Although deletion of, and point mutations within, the 7-bp TATA-like box reduced CAT synthesis to 16-50% of that obtained with the wild-type promoter, only deletion of the 7-bp sequence caused a detectable shift of the transcription start site, indicating that the function of the AT-rich region is to position the RNA polymerase. The most significant reduction in CAT synthesis--to 1.5% of wild-type--occurred after deletion of the 23-bp immediately 5' to the TATTTTA box, which marks this 23-bp sequence as the critical cis-regulatory element for FV3 trans-activation.

The primary structure of the thymidine kinase gene of fish lymphocystis disease virus.

The DNA nucleotide sequence of the thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) which has been localized between the coordinates 0.678 to 0.688 of the viral genome was determined. The analysis of the DNA nucleotide sequence located between the recognition sites of HindIII (0.669 map unit; nucleotide position 1) and AccI (nucleotide position 2032) revealed the presence of an open reading frame of 954 bp on the lower strand of this region between nucleotide positions 1868 (ATG) and 915 (TAA). It encodes for a protein of 318 amino acid residues. The evolutionary relationships of the TK gene of FLDV to the other known TK genes was investigated using the method of progressive sequence alignment. These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene and the amino acid composition of other TKs tested. However, significant conservations were detected at several regions of amino acid residues of the FLDV TK protein when compared to the amino acid sequence of TKs of African swine fever virus, fowlpox virus, shope fibroma virus, and vaccinia virus and to the amino acid sequences of the cellular cytoplasmic TK of chicken, mouse, and man.