ABSTRACT
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.
Subject(s)
Endopeptidases/metabolism , Luteovirus/enzymology , Mutation/genetics , Solanum tuberosum/virology , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution/genetics , Base Sequence , Capsid/biosynthesis , Capsid/genetics , Caulimovirus/genetics , Endopeptidases/chemistry , Endopeptidases/genetics , Genetic Complementation Test , Genome, Viral , Luteovirus/genetics , Luteovirus/physiology , Open Reading Frames/genetics , Plant Leaves/microbiology , Plant Leaves/virology , Promoter Regions, Genetic/genetics , Protein Structure, Tertiary , RNA, Viral/biosynthesis , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Rhizobium/genetics , Serine/genetics , Serine/metabolism , Solanum tuberosum/microbiology , Virus ReplicationABSTRACT
Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.
