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Journal of Virology, April 2002, p. 3461-3470, Vol. 76, No. 7
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.7.3461-3470.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains

Catherine Eichwald, Fulvia Vascotto, Elsa Fabbretti,,{dagger} and Oscar R. Burrone*

International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy

Received 10 August 2001/ Accepted 11 December 2001

Rotavirus NSP5 is a nonstructural protein that localizes in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced polyacrylamide gel electrophoresis mobility. This process has been suggested to be due in part to autophosphorylation. We developed an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA104 cells transfected with NSP5 mutants but not by extracts from mock-transfected cells. The phosphorylated products obtained showed shifts in mobility similar to what occurs in vivo. From these and other experiments we concluded that NSP5 activates a cellular kinase(s) for its own phosphorylation. Three NSP5 regions were found to be essential for kinase(s) activation. Glutathione S-transferase-NSP5 mutants were produced in Escherichia coli and used to determine phosphoacceptor sites. These were mapped to four serines (Ser153, Ser155, Ser163, and Ser165) within an acidic region with homology to casein kinase II (CKII) phosphorylation sites. CKII was able to phosphorylate NSP5 in vitro. NSP5 and its mutants fused to enhanced green fluorescent protein were used in transfection experiments followed by virus infection and allowed the determination of the domains essential for viroplasm localization in the context of virus infection.

* Corresponding author. Mailing address: International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy. Phone: 39-040-37571. Fax: 39-040-3757362. E-mail: burrone{at}icgeb.trieste.it.

{dagger} Present address: Centre for Rare Diseases, IRCCS, Ospedale Burlo Garofolo, 34137 Trieste, Italy.

Journal of Virology, April 2002, p. 3461-3470, Vol. 76, No. 7
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.7.3461-3470.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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