Abstract
Genomic analysis of Lettuce infectious yellows virus (LIYV) has revealed two short open reading frames (ORFs) on LIYV RNA2, that are predicted to encode a 5-kDa (P5) and a 9-kDa (P9) protein. The P5 ORF is part of the conserved quintuple gene block in the family Closteroviridae, while P9 orthologs are found in all Criniviruses. In this study, the expression of LIYV P5 and P9 proteins was confirmed; P5 is further characterized as an endoplasmic reticulum (ER)-localized integral transmembrane protein and P9 is a soluble protein. The knockout LIYV mutants presented reduced symptom severity and virus accumulation in Nicotiana benthamiana or lettuce plants, indicating their importance in efficient virus infection. The P5 mutant was successfully complemented by a dislocated P5 in the LIYV genome. The structural regions of P5 were tested and all were found to be required for the appropriate functions of P5. In addition, P5, as well as its ortholog P6, encoded by Citrus tristeza virus (CTV) and another ER-localized protein encoded by LIYV RNA1, were found to cause cell death when expressed in N. benthamiana plants from a TMV vector, and induce ER stress and the unfolded protein response (UPR).
Highlights
RNA viruses have evolved to compress maximal coding and regulatory information into minimal sequence space
To generate P5- and P9-GFP fusion constructs for transient expression in Nicotiana benthamiana epidermal cells, P5 and P9 sequences cloned from the Lettuce infectious yellows virus (LIYV) RNA2 infectious clone were assembled into the pEAQ-HT vector [26]
The two small open reading frames (ORFs) coding for the P5 and P9 orthologs, either conserved among all members in the family Closteroviridae (P5) or in the genus Crinivirus (P9), are predicted based on nucleotide sequence and have been featured as part of their characteristic viral genomes
Summary
RNA viruses have evolved to compress maximal coding and regulatory information into minimal sequence space. The 6-kDa viral protein 6K2 of Turnip mosaic virus (TuMV, Potyvirus) is a membrane-associated protein that induces the formation of endoplasmic reticulum (ER)-derived vesicles for viral genome amplification, and mediates their export from the ER for virus systemic infection [2,3]. Another 6-kDa potyviral protein, 6K1, is required for viral replication and targets the viral replication complex at the early stage of infection [4]. It is imperative to identify the coding regions and understand their functions to decipher how these small ORFs/proteins promote the infection cycle
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