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.

A virion-associated protein kinase induces apoptosis.

Apoptosis and inhibition of host gene expression are often associated with virus infections. Many viral polypeptides modulate apoptosis by direct interaction with highly conserved apoptotic pathways. Some viruses induce apoptosis during late stages of the infection cycle, while others inhibit apoptosis to facilitate replication or maintain persistent infection. In previous work, we showed that Chilo iridescent virus (CIV) or CIV virion protein extract induces apoptosis in spruce budworm and cotton boll weevil cell cultures. Here, we characterize the product of a CIV gene (iridovirus serine/threonine kinase; istk) with signature sequences for S/T kinase and ATP binding. ISTK appears to belong to the superfamily, vaccinia-related kinases (VRKs). The istk gene was expressed in Pichia pastoris vectors. Purified ISTK (48 kDa) exhibited S/T kinase activity. Treatment with ISTK induced apoptosis in budworm cells. A 35-kDa cleavage product of ISTK retaining key signature sequences was identified during purification. Pichia-expressed 35-kDa polypeptide, designated iridoptin, induced apoptosis and inhibition of host protein synthesis in budworm and boll weevil cells. A mutation in the ATP-binding site eliminated both kinase and apoptosis activity of iridoptin, suggesting that kinase activity is essential for induction of apoptosis. Analysis with custom antibody confirmed that ISTK is a structural component of CIV particles. This is the first demonstration of a viral kinase inducing apoptosis in any virus-host system and the first identification of a factor inducing apoptosis or host protein shutoff for the family Iridoviridae.

Identification and characterization of a novel gene of grouper iridovirus encoding a purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway. It catalyses the reversible phosphorolysis of purine (2'-deoxy)ribonucleosides to free bases and (2'-deoxy)ribose 1-phosphates. Here, a novel piscine viral PNP gene that was identified from grouper iridovirus (GIV), a causative agent of an epizootic fish disease, is reported. This putative GIV PNP gene encodes a protein of 285 aa with a predicted molecular mass of 30 332 Da and shows high similarity to the human PNP gene. Northern and Western blot analyses of GIV-infected grouper kidney (GK) cells revealed that PNP expression increased in cells with time from 6 h post-infection. Immunocytochemistry localized GIV PNP in the cytoplasm of GIV-infected host cells. PNP-EGFP fusion protein was also observed in the cytoplasm of PNP-EGFP reporter construct-transfected GK and HeLa cells. From HPLC analysis, the recombinant GIV PNP protein was shown to catalyse the reversible phosphorolysis of purine nucleosides and could accept guanosine, inosine and adenosine as substrates. In conclusion, this is the first report of a viral PNP with enzymic activity.

Insect iridescent virus type 6 encodes a polypeptide related to the largest subunit of eukaryotic RNA polymerase II.

Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of known large subunits of DdRPs contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains [RQP(T/S)LH and NADFDGDE] were used in PCR experiments for the detection of the corresponding gene of the genome of insect iridescent virus type 6, also known as Chilo iridescent virus (CIV). A specific DNA product of about 150 bp could be amplified and was used as a hybridization probe against the CIV gene library to identify the corresponding gene. The gene encoding the DdRP was identified within the EcoRI fragments M (7099 bp) and L (7400 bp) of CIV DNA, between map units 0.310 and 0.347 (7990 bp). The DNA nucleotide sequence (3153 bp) of the gene encoding the largest subunit of DdRP (RPO1) was determined. Northern blot hybridization revealed the presence of a 3.4 kb RNA transcript in CIV-infected cells that hybridized to the CIV DdRP gene. This predicted viral protein consists of 1051 amino acid residues (120K) and showed considerably higher similarity to the largest subunit of eukaryotic RNA polymerase II than to the homologous proteins of vaccinia virus and African swine fever virus. Phylogenetic analysis suggested that the putative RPO1 of CIV could have evolved from RNA polymerase II after the divergence of the three types of eukaryotic RNA polymerases. The putative RPO1 of CIV lacked the C-terminal domain that is conserved in eukaryotic, eubacterial and other viral RNA polymerases and in this respect was analogous to the RNA polymerases of Archaea. It is hypothesized that the equivalent of the C-terminal domain may reside in another subunit of CIV DdRP encoded by an unidentified viral gene.

Chilo iridescent virus encodes a putative helicase belonging to a distinct family within the "DEAD/H" superfamily: implications for the evolution of large DNA viruses.

The complete nucleotide sequence of the EcoRI DNA fragment M (7099 bp; 0.310-0.345 map units) of the genome of insect iridescent virus type 6--Chilo iridescent virus (CIV)--was determined. A 606 codon open reading frame located in this region encoded a protein (p69) related to a distinct family of putative DNA and/or RNA helicases belonging to the "DEAD/H" superfamily. Unique sequence signatures were derived that allowed selective retrieval of the putative helicases of the new family from amino acid sequence databases. The family includes yeast, Drosophila, mammalian, and bacterial proteins involved in transcription regulation and in repair of damaged DNA. It is hypothesized that p69 of CIV may be a DNA or RNA helicase possibly involved in viral transcription. A distant relationship was observed to exist between this family of helicases and another group of proteins that consists of putative helicases of poxviruses, African swine fever virus, and yeast mitochondrial plasmids. It is shown that p69 of CIV is much more closely related to cellular helicases than any of the other known viral helicases. Phylogenetic analysis suggested an independent origin for the p69 gene and the genes encoding other viral helicases.