Evidence for 4-hydroxyproline in viral proteins - Characterization of a viral prolyl 4-hydroxylase and its peptide substrates

4-Hydroxyproline, the characteristic amino acid of collagens and collagen-l ike proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins . We report here on the cloning and characterization from a eukaryotic alga l virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptid e, which shows distinct sequence similarity to the C-terminal half of the c atalytic alpha subunits of animal prolyl 4-hydroxylases. The recombinant po lypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standar d substrates of animal and plant prolyl 4-hydroxylases, respectively. Synth etic peptides such as (Pro-Ala-Pro-Lys)(n) (Ser-Pro-Lys-Pro Pro)(5) and (Pr o-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the vi ral genome also served as substrates, (Pro-Ala-Pro-Lys)(10) was a particula rly good substrate, with a K-m, of 20 mu M. The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydrox ylated more efficiently. The data strongly suggest that P. bursaria Chlorel la virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase.