Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R

Abstract

this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions. -terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala. .

Background

this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions.

Results

-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala.

Conclusions

.

Background

].

. In a mutant strain of virus, temperature-sensitive for B1R, the proportion of underphosophorylated H5R decreases at the restrictive temperature (G. Beaud and R. Beaud, unpublished).

] suggest a role in virion morphogenesis. A knowledge of the phosphorylation sites on the H5R protein is needed to test whether phosphorylation has a role in either of these processes, and we have made the first steps in this direction by identifying two threonine residues in the protein that are substrates for the B1R protein kinase.

Results

).

P was released from the peptide predominantly at cycles 4, 5 and 6. As there is always carry-over from one cycle to the next, we can conclude that both residues Thr-84 and Thr-85 of H5R are phosphorylated by the vaccinia virus B1R protein kinase. Analysis of fraction 28 gave similar results to those for fraction 29 (not shown).

-terminal side.

P.

P radioactivity released at each stage measured. The identity of the phenylthiohydantoin derivative from the first seven cycles was unequivocal and is indicated.

EKN, and lane 4; no peptide.

Discussion

].

, the peptides resolved from which were insufficiently radioactive for sequence analysis.

, and, if so, with what physiological significance. However it should now be possible to address this question by constructing recombinant virus with amino acid substitutions at the positions of phosphorylation.

Conclusions

at the threonine residues Thr-84 and Thr-85 within the region:

EKNSP

A synthetic peptide based on this sequence also acted as a substrate. We conclude that this sequence determines, at least in part, the substrate specificity of the vaccinia B1R protein kinase, although it is unclear which amino acid residues are the key determinants within this sequence. There are other phosphorylation sites for the kinase on protein H5R, but these remain to be determined.

Preparation and labelling of H5R protein

P]ATP (50 μCi) and dithiothreitol (2 mM) in a total volume of 500 μl. Incubation was for 30 min at 30° C.

Proteolytic digestion of H5R protein

The 500 μl reaction mixtures, above, were adjusted to 50 mM Tris-HCl, pH 7.4, and 0.01% reduced Triton X-100 at a final volume of 600 μl. To this was added 0.4 μg V8 protease (Boehringer Mannheim) and incubation carried out at 30° C for 18 h. To prepare the peptides for HPLC analysis the reaction mixtures were pooled and applied to a SEP-PAK cartridge (prewashed with successive 10 ml portions of 50% acetonitrile and water) and eluted with water (40 ml) followed by 50% acetonitrile (40 ml) and finally 100% acetonitrile (30 ml). A large peak of radioactivity (unreacted ATP) eluted with the water and was discarded, and a smaller broad peak of radioactivity that eluted with 50% acetonitrile was retained and concentrated to 200 μl by rotary evaporation.

Purification of H5R peptides and sequence analysis

].

Labelling and analysis of synthetic peptides

P]ATP (6.3 μCi) B1R protein kinase (4 μl), Tris-HCl, pH 7.4 (20 mM), magnesium chloride (5 mM), ATP (50 μM), and dithiothreitol (2 mM) in a total volume of 20 μl. Incubation was for 30 min at 30° C. The reaction mixtures were applied in 1 cm strips to thin layer cellulose plates and subjected to electrophoresis for 4 h at 200 V in a solution of pyridine : acetic acid : water (20:200:1780) at pH 3.5. The plates were dried, stained with ninhydrin to locate the unphosphorylated peptides, and subjected to autoradiography.

Acknowledgements

We thank the Wellcome Trust for support.