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The 3′ untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3′UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3′ cap-independent translation enhancers (3′CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3′TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3′ terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed “Trio”, that are exclusively found in a subset of umbraviruses and are located just upstream of the 3′TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3′ terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3′CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement. 

ABSTRACT Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae . OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNA D ). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3′ untranslated region of OPMV contains two 3′ cap-independent translation enhancers (3′ CITEs), a T-shaped structure (TSS) near its 3′ end, and a Barley yellow dwarf virus -like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5′ sequences in vivo , which differs from how umbravirus 3′ CITEs were used in a previous study. Similarly to most 3′ CITEs, the OPMV BTE links to the 5′ end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence. IMPORTANCE Opium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae . We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5′ end just upstream from a previously predicted xrRNA D site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNA D sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo . We also characterized the OPMV BTE structure, a 3′ cap-independent translation enhancer (3′ CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3′ CITEs.