Functional analysis and characterization of protein kinase gene (AMV197) of Amsacta moorei entomopoxvirus

Amsacta moorei Entomopoxvirus (AMEV) is an insect viruses that belongs to Poxviridae family and Entomopoxviridae subfamily, and has high level pathogenic effect on insect pests. In addition, AMEV can be used as gene expression and gene therapy vectors, and has great importance as model virus for studying the mechanism of human viruses.In this work, in order to identify the AMEV genome, transcriptional and functional analysis of AMV197 which is found in AMEV genome and has serine/threonine protein kinase domain was performed.In the presence of DNA replication inhibitor, transcriptional analyses done with mRNA from Ld652 cells infected with AMEV by using RT-PCR methods showed that AMV197 is expressed as an early group gene. Also, time dependent transcription profile of AMV197 showed that transcription of AMV197 started 4 hour after infection, increased to the high level at 7th hour and started to decrease from this hour.5? RACE analysis showed that transcription started at the -54th nucleotide relevant to translational start point. Result of 3? RACE showed that AMV197 has two stop points, one of them is located at the 21st nucleotide and the other point is at the 31st nucleotide down stream of translational stop point (TAA).For functional analysis of AMV197 gene has been deleted from AMEV genome by homolog recombination. DNA replication of constructed recombinant virus (Am?PK/gfp) was determined by slot-blot hybridization analysis, and infectivity of the recombinant virus was determined by EPDA (End Point Dilution Assay). These results showed that DNA replication occurred six hours earlier relevant to wild type virus, but recombinant virus production decreased by 61% relevant to wild type virus.