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Identification of artichoke mottled crinkle virus (AMCV) proteins required for virus replication: Complementation of AMCV p33 and p92 replication defective mutants

TitleIdentification of artichoke mottled crinkle virus (AMCV) proteins required for virus replication: Complementation of AMCV p33 and p92 replication defective mutants
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication1998
AuthorsMolinari, P., Marusic Carla, Lucioli Alessandra, Tavazza Raffaela, and Tavazza Mario
JournalJournal of General Virology
Volume79
Pagination639-647
ISSN00221317
Keywordsartichoke mottled crinkle virus, article, Base Sequence, Blotting, Cynara scolymus, genetic complementation, Genetic Complementation Test, Mutagenesis, Nicotiana benthamiana, Northern, open reading frame, Open Reading Frames, plant virus, Plants, priority journal, Protoplasts, RNA viruses, stop codon, Tobacco, Tombusvirus, Toxic, Viral Proteins, virus mutant, virus protein, virus replication, virus RNA
Abstract

Mutagenesis of the artichoke mottled crinkle virus (AMCV) genome and complementation studies between replication-defective mutants were undertaken to identify viral protein(s) essential for AMCV replication. Inoculation of Nicotiana benthamiana protoplasts with mutant transcripts revealed that null mutations in ORFs 1 [tA33(-)], 2 [tA92(-)] and 6 [tA7(-)], as well as an ORF 2 mutation [tA92GED] in the GDD motif of the 92 kDa protein, the putative replicase, prevented accumulation of detectable levels of progeny RNA. Conversely, mutations of ORFs 3 [tA41(-)], 4 [tA21(-)] and 5 [tA19(-)] did not substantially affect the accumulation of AMCV genomic and subgenomic RNAs of both positive and negative polarity. Inoculation of N. benthamiana plants with transcripts impaired in replication revealed that tA92(-) and tA7(-) mutants lead to replicating pseudorevertants. Functional analysis of these pseudorevertants showed that: (i) the double stop codon introduced at the end of ORF 1 to prevent the translational readthrough of the 92 kDa protein reverted to a single amber, ochre or opal codon, giving rise to viable genomes; (ii) the putative 7 kDa protein is not essential for genome viability, although the RNA region spanning ORF 6 plays a role in cis in replication. Finally, the two replication-defective mutants tA33(-) and tA92(-) complemented when coinoculated to N. benthamiana protoplasts, definitively proving that the 33 kDa protein is essential for tombusvirus genome replication. Analysis of viral RNAs from the coinfection experiments showed that tA92(-) was preferentially amplified over tA33(-).

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URLhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-0031905426&partnerID=40&md5=1ad7a9e082017f282293f9807abf1c75
DOI10.1099/0022-1317-79-3-639
Citation KeyMolinari1998639