Structure of grouper iridovirus purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides to the corresponding free bases and ribose 1-phosphate. The crystal structure of grouper iridovirus PNP (givPNP), corresponding to the first PNP gene to be found in a virus, was determined at 2.4 A resolution. The crystals belonged to space group R3, with unit-cell parameters a = 193.0, c = 105.6 A, and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an alpha/beta structure with a nine-stranded mixed beta-barrel flanked by a total of nine alpha-helices. The predicted phosphate-binding and ribose-binding sites are occupied by a phosphate ion and a Tris molecule, respectively. The geometrical arrangement and hydrogen-bonding patterns of the phosphate-binding site are similar to those found in the human and bovine PNP structures. The enzymatic activity assay of givPNP on various substrates revealed that givPNP can only accept 6-oxopurine nucleosides as substrates, which is also suggested by its amino-acid composition and active-site architecture. All these results suggest that givPNP is a homologue of mammalian PNPs in terms of amino-acid sequence, molecular mass, substrate specificity and overall structure, as well as in the composition of the active site.

Complete genome sequence of the grouper iridovirus and comparison of genomic organization with those of other iridoviruses.

The complete DNA sequence of grouper iridovirus (GIV) was determined using a whole-genome shotgun approach on virion DNA. The circular form genome was 139,793 bp in length with a 49% G + C content. It contained 120 predicted open reading frames (ORFs) with coding capacities ranging from 62 to 1,268 amino acids. A total of 21% (25 of 120) of GIV ORFs are conserved in the other five sequenced iridovirus genomes, including DNA replication, transcription, nucleotide metabolism, protein modification, viral structure, and virus-host interaction genes. The whole-genome nucleotide pairwise comparison showed that GIV virus was partially colinear with counterparts of previously sequenced ranaviruses (ATV and TFV). Besides, sequence analysis revealed that GIV possesses several unique features which are different from those of other complete sequenced iridovirus genomes: (i) GIV is the first ranavirus-like virus which has been sequenced completely and which infects fish other than amphibians, (ii) GIV is the only vertebrate iridovirus without CpG sequence methylation and lacking DNA methyltransferase, (iii) GIV contains a purine nucleoside phosphorylase gene which is not found in other iridoviruses or in any other viruses, (iv) GIV contains 17 sets of repeat sequence, with basic unit sizes ranging from 9 to 63 bp, dispersed throughout the whole genome. These distinctive features of GIV further extend our understanding of molecular events taking place between ranavirus and its hosts and the iridovirus evolution.

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.

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog.

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathke's pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.

Characterization of transactivation domain and developmental expression of pituitary specific transcription factor, Pit-1 of ayu (Plecoglossus altivelis).

Pit-1 is a pituitary-specific transcription factor, which regulates the expression of growth hormone, prolactin, and thyroid stimulating hormone-beta genes. We previously reported the expression of a Pit-1 gene from ayu (Plecoglossus altivelis), which is an important cultivated food fish in Taiwan and Japan. Comparison of ayu Pit-1 with that of salmon, turkey, and rodent, revealed that the Pit-1 structure is highly conserved through vertebrates, especially in POU-specific and POU-homeo domains. The variation among fish, bird, and mammal are mainly found in transactivation domain by alternative splicing and initiation. Three insertions were found. The gamma-insert in fish Pit-1 is homologous to the exon 2a of avian Pit-1, which is not found in mammals. The beta-insert of fish Pit-1 is homologous to the 28 amino acids (a.a.) and 26 a.a. insert of avian Pit-1 beta(*) and mammalian Pit-1 beta, respectively. An additional similarity was noticed between fish and bird, as both of them contain 7 a.a. insert that is not present in mammalian Pit-1. By site directed mutagenesis, we demonstrated that the beta, gamma, and the 7 a.a. inserts of ayu Pit-1 are critical for activation of zebrafish growth hormone promoter. The ayu Pit-1 protein was found to be expressed specifically in pituitary gland, and its mRNA was first detected at embryonic day 4, significantly increased at embryonic day 5, then sustained to time of hatching at day 8.