Analysis of Rotavirus Nonstructural Protein NSP5 Phosphorylation

BLACKHALL, J; MUÑOZ, M; FUENTES, A.; MAGNUSSON, G.

JOURNAL OF VIROLOGY

AMER SOC MICROBIOLOGY

Lugar: Washington; Año: 1998 vol. 72 p. 6398 - 6405

0022-538X

The rotavirus nonstructural phosphoprotein NSP5 is encoded by a gene in RNA segment 11. Immunofluorescence analysis of fixed cells showed that NSP5 polypeptides remained confined to viroplasms even at a latestage when provirions migrated from these structures. When NSP5 was expressed in COS-7 cells in the absenceof other viral proteins, it was uniformly distributed in the cytoplasm. Under these conditions, the 26-kDa polypeptide predominated. In the presence of the protein phosphatase inhibitor okadaic acid, the highly phosphorylated 28- and 32- to 35-kDa polypeptides were formed. Also, the fully phosphorylated protein had a homogeneous cytoplasmic distribution in transfected cells. In rotavirus SA11-infected cells, NSP5 synthesis was detectableat 2 h postinfection. However, the newly formed 26-kDa NSP5 was not converted to the 28- to 35-kDa formsuntil approximately 2 h later. Also, the protein kinase activity of isolated NSP5 was not detectable until the 28-and 30- to 35-kDa NSP5 forms had been formed. NSP5 immunoprecipitated from extracts of transfected COS-7cells was active in autophosphorylation in vitro, demonstrating that other viral proteins were not required forthis function. Treatment of NSP5-expressing cells with staurosporine, a broad-range protein kinase inhibitor,had only a limited negative effect on the phosphorylation of the viral polypeptide. Staurosporine did not inhibitautophosphorylation of NSP5 in vitro. Together, the data support the idea that NSP5 has an autophosphorylation activity that is positively regulated by addition of phosphate residues at some positions.