Abstract
The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the trans-activator (TA) that (i) promotes subgenomic mRNA transcription from RNA1, (ii) facilitates replication of RNA2, and (iii) mediates particle assembly and copackaging of genome segments. The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.
Highlights
Dianthovirus is the only genus in the family Tombusvididae that possesses a segmented genome and, as such, its members face unique challenges [1]
We have identified a novel RNA element in RNA1 of the segmented positive-strand RNA genome of Red clover necrotic mosaic virus (RCNMV)
Its similarity in structure to a known important RNA element in RNA2 suggests that the two RNA structures perform similar viral functions in their respective genome segments, and this was confirmed experimentally
Summary
Dianthovirus is the only genus in the family Tombusvididae that possesses a segmented genome and, as such, its members face unique challenges [1]. There are currently three approved dianthoviruses, the type species Carnation ringspot virus (CRSV), Sweet clover necrotic mosaic virus (SCNMV), and Red clover necrotic mosaic virus (RCNMV) [2,3,4]. These viruses contain plus-strand RNA genomes that are composed of two segments, which are copackaged into ~36 nm icosahedral particles. The 30-third of RNA1 encodes the coat protein (CP), which is translated from a subgenomic (sg) mRNA that is transcribed during infections [13]. RNA2 encodes only p35, which facilitates virus movement within plants [14]
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