Abstract
Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathione S-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, maribavir. Sequencing and mass spectrometric analyses of purified (32)P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P+5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P+5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.
Highlights
Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown
To investigate the specificity of this phosphorylation further, we initially tested the phosphorylation of calf thymus histone H2B in low salt (Ͻ5 mM NaCl). Under these conditions (Fig. 1A, lane 1), H2B was efficiently radiolabeled in the presence of [␥-32P]ATP and glutathione S-transferase (GST)-UL97
These results indicated that most of the phosphorylation of H2B in Fig. 1A was due to GST-UL97 and not a contaminant in the enzyme preparation
Summary
Cell and Viruses—Spodoptera frugiperda 9 cells (Sf9), obtained from the American Type Culture Collection, were maintained in Grace’s insect medium (Invitrogen) supplemented with 10% fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin, and 100 g/ml streptomycin B. Protein Kinase Assays—For phosphorylation of calf thymus histone H2B by UL97, assays were performed in 50 mM Tris-HCl (pH 9.0), 300 mM NaCl, 10 mM MgCl2, 2 mM dithiothreitol, and 100 M ATP), with 5 Ci/sample [␥-32P] ATP at Ͼ5000 Ci/mmol (ICN), unless otherwise specified, and various concentrations (determined by amino acid analysis) of calf thymus histone H2B (Roche Molecular Biochemicals; “electrophoretically homogeneous”) and selected amounts of GST-UL97, as specified. Identification of Phosphorylation Sites by Sequencing and Mass Spectrometry—The radioactive HPLC fractions were analyzed by peptide sequencing and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) by James Lee at the Molecular Biology Core Facility, Dana-Farber Cancer Institute. Sequencing was performed on an Applied Biosystems 477A system at the Molecular Biology Core Facility (Dana-Farber Cancer Institute) using standard techniques except that 90% (v/v) methanol containing 15 ml of 85% (w/v) phosphoric acid/100 ml was employed for residue extraction because this gave better recovery of phosphorylated derivatives. The radioactive peptides were excised, extracted, and analyzed by mass spectrometry and amino acid sequencing, as described above
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