Nicotiana benthamiana plants asymptomatically infected by Pelargonium line pattern virus show unusually high accumulation of viral small RNAs that is neither associated with DCL induction nor RDR6 activity.

Pelargonium line pattern virus (PLPV, Tombusviridae) normally establishes systemic, low-titered and asymptomatic infections in its hosts. This type of interaction may be largely determined by events related to RNA silencing, a major antiviral mechanism in plants. This mechanism is triggered by double or quasi double-stranded (ds) viral RNAs which are cut by DCL ribonucleases into virus small RNAs (vsRNAs). Such vsRNAs are at the core of the silencing process as they guide sequence-specific RNA degradation Host RNA dependent-RNA polymerases (RDRs), and particularly RDR6, strengthen antiviral silencing by promoting biosynthesis of secondary vsRNAs. To approach PLPV-host relationship, here we have characterized the vsRNAs that accumulate in PLPV-infected Nicotiana benthamiana. Such accumulation was found unprecedented high despite DCLs were not induced in infected tissue and neither vsRNA generation nor PLPV infection was apparently affected by RDR6 impairment. From the obtained data, triggers and host factors likely involved in anti-PLPV silencing are proposed.

Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3´-Translational Enhancer that Communicates with the Corresponding 5´-Region through a Long-Distance RNA-RNA Interaction.

Cap-independent translational enhancers (CITEs) have been identified at the 3´-terminal regions of distinct plant positive-strand RNA viruses belonging to families Tombusviridae and Luteoviridae. On the bases of their structural and/or functional requirements, at least six classes of CITEs have been defined whose distribution does not correlate with taxonomy. The so-called TED class has been relatively under-studied and its functionality only confirmed in the case of Satellite tobacco necrosis virus, a parasitic subviral agent. The 3´-untranslated region of the monopartite genome of Pelargonium line pattern virus (PLPV), the recommended type member of a tentative new genus (Pelarspovirus) in the family Tombusviridae, was predicted to contain a TED-like CITE. Similar CITEs can be anticipated in some other related viruses though none has been experimentally verified. Here, in the first place, we have performed a reassessment of the structure of the putative PLPV-TED through in silico predictions and in vitro SHAPE analysis with the full-length PLPV genome, which has indicated that the presumed TED element is larger than previously proposed. The extended conformation of the TED is strongly supported by the pattern of natural sequence variation, thus providing comparative structural evidence in support of the structural data obtained by in silico and in vitro approaches. Next, we have obtained experimental evidence demonstrating the in vivo activity of the PLPV-TED in the genomic (g) RNA, and also in the subgenomic (sg) RNA that the virus produces to express 3´-proximal genes. Besides other structural features, the results have highlighted the key role of long-distance kissing-loop interactions between the 3´-CITE and 5´-proximal hairpins for gRNA and sgRNA translation. Bioassays of CITE mutants have confirmed the importance of the identified 5´-3´ RNA communication for viral infectivity and, moreover, have underlined the strong evolutionary constraints that may operate on genome stretches with both regulatory and coding functions.

Evidence supporting a premature termination mechanism for subgenomic RNA transcription in Pelargonium line pattern virus: identification of a critical long-range RNA-RNA interaction and functional variants through mutagenesis.

Pelargonium line pattern virus (PLPV) is a plus-strand RNA virus that has been proposed as type species of a tentative new genus, Pelarspovirus, in the family Tombusviridae. One of the singular traits of members of this prospective genus is the production of a unique subgenomic (sg) mRNA that is structurally and functionally tricistronic. Here, we have aimed to get insights into the mechanism that governs PLPV sg mRNA transcription. A long-range RNA-RNA interaction that is critical for the process has been identified through RNA folding predictions and mutational analysis of the viral genome. Such interaction seems to occur in the plus-strand, likely acts in cis, and specifically mediates the synthesis of sg RNA-sized minus-strand. The accumulation of this RNA species is easily detectable in plants and its generation can be uncoupled from that of the plus-strand sg mRNA. All these data together with the observation that 5' ends of PLPV genomic and sg mRNAs have sequence resemblances (as expected if both act as promoters in the corresponding minus-strand), support that premature termination is the mechanism underlying PLPV sg mRNA formation.