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Journal of Virology, August 2004, p. 7945-7957, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.7945-7957.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Assembly of Turnip Yellow Mosaic Virus Replication Complexes: Interaction between the Proteinase and Polymerase Domains of the Replication Proteins

Anna Jakubiec,¹ Julien Notaise,¹ Vincent Tournier,¹ François Héricourt,^1, Maryse A. Block,² Gabrièle Drugeon,¹ Linda van Aelst,³ and Isabelle Jupin^1*

Laboratoire de Virologie Moléculaire, Institut Jacques Monod, UMR 7592, CNRS-Universités Paris 6-Paris 7, 75251 Paris Cedex 05,¹ Laboratoire de Physiologie Cellulaire Végétale, UMR 5168, CNRS-CEA-Université Joseph Fourier-INRA, CEA-Grenoble, DRDC/PCV, 38054 Grenoble Cedex 9, France,² Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724³

Received 13 January 2004/ Accepted 27 March 2004

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like supergroup, encodes two nonstructural replication proteins (140K and 66K), both of which are required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase activities, while the 66K protein encompasses the RNA-dependent RNA polymerase domain. Recruitment of the 66K protein to the sites of viral replication, located at the periphery of chloroplasts, is dependent upon the expression of the 140K protein. Using antibodies raised against the 140K and 66K proteins and confocal microscopy, we report the colocalization of the TYMV replication proteins at the periphery of chloroplasts in transfected or infected cells. The replication proteins cofractionated in functional replication complexes or with purified chloroplast envelope membranes prepared from infected plants. Using a two-hybrid system and coimmunoprecipitation experiments, we also provide evidence for a physical interaction of the TYMV replication proteins. In contrast to what has been found for other members of the alphavirus-like supergroup, the interaction domains were mapped to the proteinase domain of the 140K protein and to a large region encompassing the core polymerase domain within the 66K protein. Coexpression and colocalization experiments confirmed that the helicase domain of the 140K protein is unnecessary for the proper recruitment of the 66K protein to the chloroplast envelope, while the proteinase domain appears to be essential for that process. These results support a novel model for the interaction of TYMV replication proteins and suggest that viruses in the alphavirus-like supergroup may have selected different pathways to assemble their replication complexes.

Present address: Institut de Recherche sur la Biologie de l'Insecte, UMR 6035, CNRS-Université de Tours, 37200 Tours, France.

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