Mutational analysis of the proteinase function of Potato leafroll virus.
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.
Sadowy
Ewa
E
Institut des Sciences Végétales CNRS, Av. de la Terrasse, 91 198 Gif-sur-Yvette, France2.|Institute of Biochemistry and Biophysics PAS, Ul. Pawinskiego 5A, 02-106 Warsaw, Poland1.
Juszczuk
Marek
M
Institute of Biochemistry and Biophysics PAS, Ul. Pawinskiego 5A, 02-106 Warsaw, Poland1.
David
Chantal
C
Institut des Sciences Végétales CNRS, Av. de la Terrasse, 91 198 Gif-sur-Yvette, France2.
Gronenborn
Bruno
B
Institut des Sciences Végétales CNRS, Av. de la Terrasse, 91 198 Gif-sur-Yvette, France2.
Hulanicka
M Danuta
MD
Institute of Biochemistry and Biophysics PAS, Ul. Pawinskiego 5A, 02-106 Warsaw, Poland1.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D017796:Caulimovirus / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D016679:Genome, Viral; D017793:Luteovirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D009154:Mutation / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D018515:Plant Leaves / Q000382:microbiology / Q000821:virology; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism; D011198:Solanum tuberosum / Q000382:microbiology / Q000821:virology*; D014779:Virus Replication
D012367:RNA, Viral; D012694:Serine; D012324:RNA-Dependent RNA Polymerase; D010450:Endopeptidases