Identification, characterization and functional studies of serine/threonine kinase of spodoptera litura nucleopolyhedrovirus - I (SpltNPV-I)

Abstract

An open reading frame (ORF) of 819 nt to code for a 272 amino acids protein is identified in the genome of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I). Nucleotide and nucleotide sequence derived amino acid sequence analysis of this ORF suggested it to be a eukaryotic type protein kinase having conserved I-XI subdomains of Hanks kinase. In addition to kinase catalytic domains, this putative protein has two bromo-domains which could play regulatory role in transcription. The ORF expressed as ~31 kDa apoprotein in E. coli and ~33 kDa glycoprotein in Sf9 cells, the expressed protein is designated as SpltNPV-I pk1 or pk1. The protein is localized in the nucleus of the SpltNPV-I infected permissive cell line NIV-HA-197. The recombinant protein has auto-phosphorylation and substrate phosphorylation (MBP and Histone H1) activities in presence of Mn+2 or Mg+2, and these activities are inhibited by staurosporine. Mutation of Lys-50 to Met but not Lys-44 to Gln of the protein abolished its kinase activity. Kinetics of pk1 showed that rate of phosphorylation of SpltNPV-I pk1gt MBPgt Histone H1, and both MBP and Histone H1 have the Km of 3 µM. Analysis of phosphorylated protein showed the phosphorylation of serine and threonine residues but not tyrosine. All these results suggested that identified SpltNPV-I ORF codes for a serine/threonine kinase. Polyhedrin (polh) and p10 are the two hyper-expressed very late genes of nucleopolyhedroviruses. Alpha amanitin resistant transcription from SpltNPV-I polh promoter occurred with virus infected nuclear extract of NIV-HA-197 cells but not with that from uninfected nuclear extract. Anti-pk1 antibody inhibited the transcription and the inhibition reversed on addition of pk1, however, pk1 mutant protein, K50M having no phosphorylation activity did not overcome the transcription inhibition.