TRANSLATION AND MOVEMENT OF AN INFECTIOUS UMBRAVIRUS-LIKE RNA CITRUS YELLOW VEIN ASSOCIATED VIRUS

Abstract

While the citrus yellow vein disease was first reported more than 60 years ago, its causal agent, citrus yellow vein associated virus (CYVaV), was only sequenced in 2013. CYVaV was recently structurally characterized and phylogenetically classified as a Class 2 umbravirus-like associated RNA (ulaRNA), a new category of coat-protein dependent subviral RNA replicons. There is a dearth of structural and biological characterizations of ulaRNAs as well as studies on their translation regulation. CYVaV has a limited genome size (2.7 kb), and contains only two ORFs that encode replicase proteins p21 and p81. Here I show that CYVaV transcripts are infectious in Arabidopsis protoplasts and Nicotiana benthamiana plants, and CYVaV can systemically infect the latter using agro-infiltration, despite the absence of encoded movement proteins or silencing suppressors. Fluorescent in situ hybridization (FISH) revealed that CYVaV is phloem-limited, and restricted to sieve elements, companion cells, and phloem parenchyma cells. In this work, the secondary structures of the CYVaV 5ʹ end and 3ʹ UTR were determined using SHAPE structure probing and phylogenic comparisons, and were used to infer the putative structures of other Class 2 ulaRNAs, revealing a number of distinctive structural features. Here I report the identification of a novel 3ʹCITE in the 3ʹUTR of CYVaV that is strongly conserved in Class 2 ulaRNAs and structurally resembles an I-shaped structure (ISS) 3ʹCITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5ʹ end. We also report that the ~30 nt 5ʹ terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3ʹCITE, or the 3ʹCITEs of umbravirus PEMV2, or even independent of a 3ʹCITE. These findings introduce a new type of 3ʹCITE and provide the first information on translation of ulaRNAs.