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A new gene in the Luteoviridae essential for systemic infection
W. A. MILLER (1), E. Smirnova (2), A. E. Firth (3), D. Scheidecker (2), A. Rakotondrafara (4), B. Chung (3), V. Ziegler-Graff (2). (1) Iowa State Univ, Ames, IA, U.S.A.; (2) Institut de Biologie Moléculaire des Plantes, Strasbourg, France; (3) University of Cambridge, Cambridge, United Kingdom; (4) University of Wisconsin, Madison, WI, U.S.A.

The Luteoviridae family includes many important plus strand RNA viruses, e.g. Barley yellow dwarf, Potato leafroll, and Turnip yellows (TuYV) viruses. These viruses generate a subgenomic mRNA (sgRNA1) for translation of (i) the coat protein (CP), (ii) a C-terminally extended form of the CP (CP-RTD), and (iii) open reading frame 4 (ORF4). ORF4 is nested within the CP ORF in a different reading frame. Computational analysis of >200 Luteovirid genomes revealed a new ORF (ORF3a) in what was thought to be the 5’ untranslated region of sgRNA1. ORF3a (i) contains about 50 codons, (ii) lacks an AUG start codon, (iii) is in a different reading frame from either the CP ORF or ORF4, and (iv) terminates between the CP ORF start codon and the ORF 4 start codon. In vitro translation showed that ORF3a of TuYV initiates at an ACG codon. Changing this codon to AUG or AGC greatly increased or eliminated translation of ORF3a, respectively. Immunoblots detected the protein product of ORF3a (P3a) in infected cells. Previous studies of deletion mutants unwittingly showed that ORF3a is unnecessary for virus replication in single cells. However, Agroinfection with TuYV containing the above mutations in ORF3a start codons showed that ORF3a is required for viral systemic movement in plants and that overexpression of ORF3a can increase symptoms in inoculated leaves of <i>Nicotiana benthamiana</i>. Thus, P3a is a new participant in the long distance spread of luteoviruses within their host.

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