AMV153 IS ESSENTIAL FOR REPLICATION OF AMSACTA MOOREI ENTOMOPOXVIRUS (AMEV), AND PROTEIN KINASE ENCODED BY AMV197 CAN NOT COMPLETE ITS FUNCTION

The Amsacta moorei entomopoxvirus (AMEV) belongs to the Entomopoxvirinae subfamily of poxviruses. Although entomopoxviruses are very similar to poxviruses of humans, they have no potential risk for humans because they only infect insects. Intensive studies have been carried out on this virus because it can potentially be used as gene therapy and gene expression vectors. The AMEV genome has 256 open reading frames and two of them, AMV153 and AMV197, are putative protein kinases. Protein kinases of some vertebrate poxviruses have some functions in virus morphogenesis, regulation of the cell cycle, and apoptosis of the host. In previous studies, the molecular structure of AMV197 protein kinase was elucidated, its transcriptomic properties in cell culture and its function in progeny virus production were determined, and an AMV197-defective virus (AmΔPK/gfp) was produced. However, the function of AMV153 in this scenario remained as unclear, and it is crucial to know its function to better understand the role of protein kinases in the replication of AMEV. The AMV153 was first deleted from the genome of AmΔPK/gfp and subsequently from the genome of AMEV, creating two separate recombinant viruses. The homologous recombination method was used to replace AMV153 ORF with mCherry gene that produces red fluorescent protein. Mutant viruses in which the AMV153 gene was replaced with mCherry were identified by fluorescence microscopy but could not be propagated separately from the AmΔPK/gfp or wild-type viruses in insect cells. Unsuccessful attempts to isolate the mutant viruses with the AMV153 gene deletion first in AmΔPK/gfp virus and second in wild type AMEV structure, suggested that the protein kinase encoded by AMV197 cannot substitute the function of AMV153 and the AMV153 protein is essential for virus replication.