Purification and characterization of the protein kinase encoded by the UL13 gene of herpes simplex virus type 2.
The proteins encoded by the UL13 genes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) have been predicted to be protein kinases. To identify the UL13 gene product, we have raised a rabbit polyclonal antiserum against a His.Tag-HSV-1 UL13 fusion protein. The antibody specifically reacted with the 60-kDa UL13 fusion protein expressed in Escherichia coli and also recognized 56- to 57-kDa late proteins in nuclear fractions of HSV-1- and HSV-2-infected cells. On the other hand, novel casein kinase activity was induced at the late stage of infection when Vero cells were infected with HSV-1 and HSV-2. The induction of the activity was most prominent in the nuclear fractions of HSV-2-infected cells and therefore we purified the protein kinase (PK) from the nuclear extracts by successive column chromatography (phosphocellulose, DEAE-cellulose, and hydroxyapatite) using casein as an exogenous substrate. The final preparation of the enzyme contained a single major protein with an apparent molecular weight of 56 kDa which was specifically reacted with the UL13 antiserum. The PK activity was optimal in the absence of NaCl and at relatively high pH. Acidic proteins such as casein and phosvitin were efficiently phosphorylated by the PK. A basic protein, protamine, which is the best substrate for the HSV-2 US3 PK, was not detectably phosphorylated but histone was a relatively good substrate for the UL13 PK. Phosphoamino acid analysis revealed that the PK phosphorylated serine and threonine but not tyrosine. Moreover the enzyme was found to be highly resistant to heparin, a potent inhibitor of casein kinase II (CK II) and also resistant to CK I-7, a synthetic inhibitor of CK I, but very sensitive to a bioflavonoid quercetin. These results indicate that the HSV-2 UL13 PK had unique catalytic properties different from those of cellular CK I, CK II, and the viral PK encoded by the US3 gene. We have also determined the complete nucleotide sequence of the HSV-2 UL13 gene. The overall amino acid homology between the HSV-2 and HSV-1 UL13 PKs was 85.9% and the homology was highly conserved in the C-terminal region.
Daikoku
T
T
Laboratory of Virology, Nagoya University School of Medicine, Japan. daikokut@tsuru.med.nagoya-u.ac.jp
Shibata
S
S
Goshima
F
F
Oshima
S
S
Tsurumi
T
T
Yamada
H
H
Yamashita
Y
Y
Nishiyama
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D017886:Durapatite; D004790:Enzyme Induction; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018139:Simplexvirus / Q000201:enzymology*; D013379:Substrate Specificity; D014709:Vero Cells
D000906:Antibodies; D002364:Caseins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D017886:Durapatite; D011494:Protein Kinases; C082380:UL13 protein, Simplexvirus