Endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein

• Published in: FEBS letters Vol. 282; no. 2; pp. 419 - 424
• Main Authors: Mohandas, Devaki V., Dales, Samuel
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 06.05.1991; Elsevier; John Wiley and Sons Inc
• Subjects: Acid Phosphatase - metabolism; Alkaline Phosphatase - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Brain - enzymology; Capsid - metabolism; Cations, Divalent - pharmacology; Cell Compartmentation; Coronavirus; EDTA; EGTA; Endosome; Endosomes - enzymology; Enzymes and enzyme inhibitors; Ethers, Cyclic - pharmacology; Ethylenediaminetetraacetate, disodium salt; Ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; Full‐length; Fundamental and applied biological sciences. Psychology; Hydrolases; In Vitro Techniques; Mice; Murine hepatitis virus - metabolism; Neurons - enzymology; Nucleocapsid protein; Okadaic Acid; p-nitrophenyl phosphate; phenyl methyl sulfonyl fluoride; Phosphoprotein phosphatase; Phosphoprotein Phosphatases - metabolism; PMSF; pNPP; Sodium Fluoride - pharmacology; Subcellular Fractions - enzymology; Viral Core Proteins - metabolism; Coronavirus; Phosphoprotein phosphatase; PMSF; pNPP; Nucleocapsid protein; EDTA; Endosome; EGTA; Virus; Proteins; Enzymatic activity; Nucleocapside; Cell nucleus; Dephosphorylation; Coronaviridae
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(91)80528-B

On the assumption that dephosphorylation of the neurotropic coronavirus JHM (JHMV) nucleocapsid protein (N) may be connected with initiation of the infectious cycle we searched for a relevant host enzyme activity. Analysis of subcellular fractions from L-2 murine fibroblasts, separated by dual Percoll density gradients, revealed the presence of a phosphoprotein phosphatase (PPPase), co-sedimenting with the endososomal/prelysosomal material, which possesses high activity against N. With purified [ 22P]N as substrate it was demonstrated that this PPPase, distinguishable from acid and alkaline phosphatases, acts optimally at neutral pH in the presence of Mn 2+ following treatment with a detergent. Complete inhibition with okadaic acid at 0.9–4.5 μM but not at 1–10 nM relegates this PPase to a type I protein phosphatase. Similar PPPase activity for N was present in the endosome fraction of a rat Roc-1 astrocytoma-oligodendrocyte cell line and in homogenates of brain and cultured oligodendrocytes. Our data suggest that the phosphorylated N of the inoculum may be modified by the endosomal PPPase in host cells, including those from the CNS so as to facilitate the JHMV infectious process.