Abstract
Background Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5′-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability.Methodology/Principal FindingsHere we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host.Conclusions/SignificanceOur findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism.We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany.
Highlights
Ribosome shunt is a mechanism of eukaryotic translation initiation that combines features of both 59-end dependent scanning and internal ribosome entry
The Rice tungro bacilliform virus (RTBV) shunt elements imbedded in the Cauliflower mosaic virus (CaMV) leader confer efficient shunting and TAV-activated polycistronic translation in protoplasts derived from a CaMV host plant Using a PCR ligation method, we replaced two distant regions of the 612 nt CaMV leader sequence with corresponding regions of the RTBV leader (Fig. 1A)
To test the effect of this replacement on leader-controlled translation we used a transient expression system based on plant protoplasts from cell suspension of Orychophragmus violaceus, in which the CAT reporter gene is expressed from transiently-transfected plasmid constructs as part of the modified CaMV 35S RNA transcription unit
Summary
Ribosome shunt is a mechanism of eukaryotic translation initiation that combines features of both 59-end dependent scanning and internal ribosome entry It has been discovered in plants, first for Cauliflower mosaic virus (CaMV) [1,2] and for Rice tungro bacilliform virus (RTBV) [3]. Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability
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