Evidence for 4-Hydroxyproline in Viral Proteins

Abstract

4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial pro- teins. We report here on the cloning and characteriza- tion from a eukaryotic algal virus, Paramecium bur- saria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-ter- minal half of the catalytic ␣ subunits of animal prolyl 4-hydroxylases. The recombinant polypeptide, ex- pressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)10 and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively. Synthetic pep- tides such as (Pro-Ala-Pro-Lys)n, (Ser-Pro-Lys-Pro- Pro)5, and (Pro-Glu-Pro-Pro-Ala)5 corresponding to pro- line-rich repeats coded by the viral genome also served as substrates. (Pro-Ala-Pro-Lys)10 was a particularly good substrate, with a Km of 20 ?M. The prolines in both positions in this repeat were hydroxylated, those pre- ceding the alanines being hydroxylated more efficiently. The data strongly suggest that P. bursaria Chlorella virus-1 expresses proteins in which many prolines be- come hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase. 4-Hydroxyproline