Adenosine triphosphatase activity and cell growth promotion of Andrias davidianus ranavirus 96L-encoded protein (ADRV-96L)

Abstract:

[Background] Adenosine triphosphatase (ATPase) controls DNA replication initiation and host response switch to pathogenic microbes. Recent genomics studies on aquatic animal iridoviruses have suggested that they share a gene encoding ATPase. Here, the function of the core gene in iridovirus will be first elucidated. [Objective] Andrias davidianus ranavirus (ADRV), which belong to the family Iridoviridae, is the viral pathogen of mass mortality in Chinese giant salamander—the world’s largest living amphibians. In order to identify virus gene and test the gene expression effects on virus replication and host cell, an ADRV gene, ADRV-96L was cloned, expressed and function analyzed. [Methods] Sequence data were analyzed using PredicProtein software. pET32a/His ADRV-96L recombinant prokaryotic expression plasmid was constructed, and the protein was expressed in Escherichia coli DE3 using IPTG induction, purified by Nickel Resin and eluted with imidazole. ATPase activity of the purified ADRV-96L recombinant protein was measured by the production of inorganic phosphorus (Pi) using molybdenum blue spectrophotometry method. Stable transfected cells were constructed and characterized. Then, the effects of ADRV-96L were analyzed by one-step multiplication curve for virus replication and estimating the growth rates of transferred cell, respectively. [Results] The multiple sequence alignments provided that ADRV-96L was characterized by the presence of the conserved AAA-ATPase (The ATPase associated with a variety of cellular activities) domains (20–159 residues) that contain Walker A and Walker B motifs, and two highly conserved arginines. The recombinant prokaryotic plasmid expressed a 52 kD fusion protein containing ADRV-96L, and the protein has ATPase activity 4.68 U/mg. The results show that ADRV-96L has no measurable impact on the ADRV replication, but it has been shown to promote cell growth. [Conclusion] Andrias davidianus ranavirus 96L gene (ADRV-96L) encodes an ATPase that is involved in cell proliferation and growth.