Rotavirus NSP5 associated with protein kinase

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

Jerusalem

Congreso; Xth International Congress of Virology; 1996

Rotavirus NSP5 associated with protein kinaseJ. Blackhall, A. Fuentes, M. Muñoz, and G. MagnussonDepartment of medical immunology and microbiology. Uppsala University, Box 582 S-751 23 Uppsala SwedenThe nonstructural NSP5 phosphoprotein is the product of the smallest rotavirus genomic RNA segment. In infected cells NSP5 immunofluorescence accumulated in the viroplasm, co-localizing with VP6 polypeptide. Pulse and pulse-chase labelling experiments showed that the synthesis of the 26 kDa form of NSP5 was maximal at 4 h post infection and that processing by glycosylation to the 28 kDa form was maximal about 4 h later. Incubation of NSP5 polypeptide immunprecipitated from infected cells with [γ32P]ATP revealed a protein kinase activity. The 28 kDa form of NSP5 was more heavily phosphorylated in vivo and in vitro by the associated protein kinase than the 26 kDa polypeptide. This result suggests a coupling between the two processes. Moreover, the NSP5 protein transiently expressed in uninfected COS-7 cells was not converted from the 26 to the 28 kDa form and lacked the in vitro phosphorylating activity. Thus the presence of other viral or viral induced proteins was needed for the posttranslational modification that caused the shift in size. In contrast, phosphorylation of NSP5 took place in absence of other viral proteins. NSP5 expressed in transfected COS-7 cells was phosphorylated in vivo and even a GST-NSP5 fusion protein expressed in bacteria was phosphorylated in vitro when supplemented with cytosolic proteins.