Molecular studies of Chlorella virus DNA and protein modifications

Chlorella virus SC-1A encodes at least seven genes related to DNA restriction-modification, including four sequence-specific $\rm N\sp6$-adenine methyltransferases (M.CviSI-SIV), one cytosine methyltransferase (M.CviSV), one endonuclease (R.CviSIII), and one methyltransferase pseudogene. Genes encoding the three methyltransferases, M.CviSI, M.CviSII, and M.CviSIII, were cloned, sequenced, and mapped. The three methyltransferase genes were scattered throughout SC-1A genome. The predicted amino acid sequences of M.CviSI (TGC$\sp{\rm m}$A) and M.CviSIII (TCG$\sp{\rm m}$A) were compared to their isomethylomers; some important sequence motifs are conserved among Chlorella isomethylomers. The sequences of M.CviSII (C$\sp{\rm m}$ATG) and its promoter region were almost identical to those of its isomethylomer M.CviAII (C$\sp{\rm m}$ATG) from virus PBCV-1. Sequencing of a SC-1A genomic fragment revealed the presence of a gene with strong homology with eukaryotic serine/threonine specific protein kinases. The homologous gene from virus PBCV-1 was also cloned and sequenced. The predicted amino acid sequence of the protein kinases from PBCV-1 and SC-1A has 94% identity. Both Chlorella protein kinases contain all the highly conserved motifs present in all eukaryotic serine/threonine specific protein kinases. The Chlorella virus protein kinase was overexpressed in E.coli. Protein kinase activity was restored from the denatured inclusion body by in vitro folding. Both SC-1A and PBCV-1 package protein kinase activity(ies) into their virion. Chlorella virus PBCV-1 contains three glycoproteins, the major capsid protein Vp54 and two minor proteins Vp280 and Vp260. The oligosaccharide(s) are apparently attached via an O-linkage. Vp54, along with two other viral proteins (Vp51 and Vp27.5), are labeled with myristic acid via an amide linkage. The carboxyl-terminal 33 kDa cyanogen bromide cleavage fragment of Vp54 contains both myristylation and glycosylation sites. The putative gene encoding PBCV-1 and its serotype EPA-1 glycoprotein 2 was cloned and sequenced; Vp260 contains 15 similar repeats of 61 to 65 amino acids. The EPA-1 protein lacked four of the amino acid repeats and contains a duplication of one of the repeat sequences