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Mutational analysis of the proteinase function of Potato leafroll virus

Institute of Biochemistry and Biophysics PAS, Ul. Pawinskiego 5A, 02-106 Warsaw, Poland¹
Institut des Sciences Végétales CNRS, Av. de la Terrasse, 91 198 Gif-sur-Yvette, France²

Author for correspondence: Ewa Sadowy. Present address: Sera & Vaccine Central Research Laboratory, Ul. Chemska 30/34, 00-725 Warszawa, Poland. Fax +48 22 841 29 49. e-mail sadowy{at}ibbrain.ibb.waw.pl

cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513–515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.

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