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J. Biol. Chem., Vol. 281, Issue 30, 21236-21249, July 28, 2006

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Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus^*

Anna Jakubiec¹, Vincent Tournier, Gabrièle Drugeon, Stéphanie Pflieger, Laurent Camborde, Joëlle Vinh, François Héricourt¹², Virginie Redeker³, and Isabelle Jupin⁴

From the Institut Jacques Monod, 75251 Paris, France and Ecole Supérieure de Physique et Chimie Industrielles de la Ville de Paris, 75005 Paris, France

Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.

Received for publication, January 3, 2006 , and in revised form, May 17, 2006.

^* This work was supported in part by grants from the MENRT and CNRS, Actions Concertées Incitatives "Jeunes Chercheurs," "Programme de Microbiologie Fondamentale," and "Biologie Moléculaire, Cellulaire et Structurale" (to I. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a fellowship from the Ministère de l'Education Nationale de la Recherche et de la Technologie.

² Present address: Laboratoire de Biologie des Ligneux et Grandes Cultures, Université d'Orléans, 45067 Orléans, France.

³ Present address: Laboratoire d'Enzymologie et de Biochimie Structurales, UPR 9063 CNRS, 91198 Gif-sur-Yvette, France.

⁴ To whom correspondence should be addressed: Molecular Virology, Institut Jacques Monod, UMR 7592 CNRS-Universités Paris 6-Paris 7, 2 Place Jussieu, 75251 Paris, France. Tel.: 33-1-44274099; Fax: 33-1-44275716; E-mail: jupin{at}ccr.jussieu.fr.

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This article has been cited by other articles:

				A. Jakubiec, G. Drugeon, L. Camborde, and I. Jupin Proteolytic Processing of Turnip Yellow Mosaic Virus Replication Proteins and Functional Impact on Infectivity J. Virol., October 15, 2007; 81(20): 11402 - 11412. [Abstract] [Full Text] [PDF]