Conserved Structure Associated with Different 3'CITEs Is Important for Translation of Umbraviruses

The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3' structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3' cap-independent translation enhancers (3'CITEs), as umbraviruses can have different 3'CITEs in the central region of their lengthy 3'UTRs, and most also have a particular 3'CITE (the T-shaped structure or 3'TSS) near their 3' ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3'CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3'CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3'CITEs that differentially affect the structure and translation of different umbraviruses.