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Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Regulated processing of nepovirus polyproteins allows the release of mature proteins and intermediate polyproteins. Infectious cDNA clones of the mild NW isolate of arabis mosaic virus (ArMV) and chimeric clones incorporating RNA1 segments of Lv, a severe isolate, were generated. Clones containing the Lv X2-NTB cleavage site were not infectious unless the Lv protease was present. The Lv and NW X2-NTB cleavage sites differ at positions P6, P4 and P2. In vitro, processing at the X2-NTB site was undetectable or reduced in chimeric polyproteins containing the Lv X2-NTB site and the NW protease but was restored when both the Lv protease and X2-NTB site were present. In contrast, cleavage at this site was increased in polyproteins that contained the NW X2-NTB site and the Lv protease. These results show that the ArMV-Lv protease has greater activity and is active on a greater range of cleavage sites than that of ArMV-NW., (© 2013 Published by Elsevier Inc.)