Abstract
Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family.
Highlights
RNA viruses utilize a vast array of coding and gene expression strategies to maximize the utility and regulation of their genomes [1,2,3,4]
A similar functional correlation was observed when the same mutant genomes were transfected into plant protoplasts and viral genome accumulation was monitored by Northern blotting (Figure 2C). These results indicate that a long-range base pairing interaction between the proximal readthrough element (PRTE) and distal readthrough element (DRTE) is required for both efficient RT in vitro and robust genome accumulation in vivo
We provide evidence that the tombusvirus Carnation Italian ringspot virus (CIRV) utilizes two sets of long-range RNARNA interactions and an RNA switch that are functionally linked to RNA replication elements to modulate and coordinate RT and viral genome replication
Summary
RNA viruses utilize a vast array of coding and gene expression strategies to maximize the utility and regulation of their genomes [1,2,3,4]. Our results show that control of the readthrough process is complex and involves both local structures and long-range interactions within the viral genome This system is integrated with viral RNA replication elements and this allows the virus to coordinate polymerase production with genome replication. Additional long-range RNA-RNA interactions have been identified in tombusvirus genomes that mediate other important viral processes such as, transcription of viral subgenomic (sg) mRNAs that template translation of 3’-proximal viral proteins [21,22,23] or assembly of the RNA replicase complex that replicates the viral genome [24] In the latter case, two essential RNA replication elements, region II (RII) in the p95 RT region [25,26] and RIV in the 3’UTR [27,28,29], are united by an upstream linker-downstream linker (UL-DL) RNA-RNA base pairing interaction to create an RII-RIV RNA platform for replicase complex assembly [24] (Figure 1A). Components of this mechanism are conserved in other members of Tombusviridae and related viruses
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