Archivio Journal of Plant Pathology

Abstract:

Grapevine leafroll-associated virus 2 (GLRaV-2) was purified from Nicotiana benthamiana. The molecular mass of viral coat protein subunits determined by polyacrylamide gel electrophoresis was ca 21.5 kDa. Double- stranded RNA was isolated from infected N. benthamiana and used for cloning and sequencing. Molecular probes and primers generated during cloning were successfully used for virus detection in infected grapevines by dot spot hybridization and reverse-transcription polymerase chain reaction. The sequence of the 3’-terminal 8590 nucleotides of the viral genome was determined, encompassing eight open reading frames (ORFs). The first ORF consisted of two parts, ORF1a, which was incompletetely sequenced and contained the conserved domains of virus helicases in its 3’ region, and ORF1b, whose product (RNA dependent RNA polymerase) is apparently expressed via a +1 ribosomal frameshift. The ORFs that followed in the 5’-3’ direction, encoded proteins of 6 kDa (ORF2); 65 kDa (ORF3), identified as a homologue of cell heat shock proteins; 63 kDa (ORF4); 25 kDa (ORF5), identified as a diverged copy of the coat protein (CP); 22 kDa (ORF6) identified as the CP; 19 kDa (ORF7) and 24 kDa (ORF8). The structural organization of the genome was virtually identical to that of beet yellows virus , the type species of the genus Closterovirus, and also resembled that of citrus tristeza virus. This similarity was confirmed by comparative analysis of phylogenetically relevant proteins. GLRaV-2 has morphological, physicochemical, ultrastructural, and molecular properties that qualify it as a species in the genus Closterovirus.