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Polerovirus suppression by a 5’ sequence-specific RNA-binding protein encoded by a late infection 3’ subgenomic RNA

Kalischuk, Melanie and Lawrence Kawchuk (2016) Polerovirus suppression by a 5’ sequence-specific RNA-binding protein encoded by a late infection 3’ subgenomic RNA. Proceedings of RNA 2016, The 21st Annual Meeting of the RNA Society. Abstract 868. June 28 - July 2, 2016, Kyoto, Japan. (Poster) http://www2.convention.co.jp/rna2016/index.html

Abstract

Potato leafroll virus (PLRV) causes significant yield and quality losses to infected potato (Solanum tuberosum L) world-wide. Through small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs, we discovered a sgRNA ~500 nt from the 3’ terminus of PLRV genome. To characterize the role of a third sgRNA (sgRNA3) of PLRV, through svRNA analyses and in planta complementation studies with the PLRV 5’ sequence-specific RNA-binding protein encoded by the 3’ subgenomic. PLRV-infected and the healthy potato plants were maintained in sterile tissue culture and grown at 20 °C and 16 hrs photoperiod. PLRV-derived sRNA sequences were identified using the Genbank BLAST program against the PLRV Canadian isolate genomic sequence (accession number D13954.1). The PLRV-derived sRNAs covered the viral genome in near saturation with the exception of gaps that aligned to regions of the transcriptional start sites for sgRNA1, sgRNA2, and sgRNA3 and the PLRV IRES that allows for ribosomal frameshifting for expression of viral RNA dependent RNA polymerase ORF1/ORF2 and translation of the replication associated Rap1. The construction of chimeric PLRV ORF7 and ORF7-GFP vectors for transient and stable expression experiments were verified with DNA sequencing, transformed into A. tumefaciens, and expression examined in planta. To test for sequence-specific nucleic acid-binding activity of ORF7 fusion proteins were performed using filter-binding experiments. The binding activity of pGEX:ORF7 fusion proteins was observed against a specific 5’ sequence of PLRV close to the IRES . This is the first sgRNA3 identified in a virus from the genus Polerovirus and it exhibits several characteristics that suggest a regulatory role in virus replication with the 3’ sgRNA3 protein binding the 5’ PLRV gRNA replicase IRES. Expression of P7 in planta significantly reduced virus accumulation. This study advances our understanding of virus regulation and provides an innovative strategy for virus eradication.

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