Abstract
Many positive-sense RNA viruses transcribe subgenomic (sg) mRNAs during infections that template the translation of a subset of viral proteins. Red clover necrotic mosaic virus (RCNMV) expresses its capsid protein through the transcription of a sg mRNA from RNA1 genome segment. This transcription event is activated by an RNA structure formed by base pairing between a trans-activator (TA) in RNA2 and a trans-activator binding site (TABS) in RNA1. In this study, the impact of the structural context of the TABS in RNA1 on the TA–TABS interaction and sg mRNA transcription was investigated using in vitro and in vivo approaches. The results (i) generated RNA secondary structure models for the TA and TABS, (ii) revealed that the TABS is partially base paired with proximal upstream sequences, which limits TA access, (iii) demonstrated that the aforementioned intra-RNA1 base pairing involving the TABS modulates the TA–TABS interaction in vitro and sg mRNA levels during infections, and (iv) revealed that the TABS in RNA1 can be modified to mediate sg mRNA transcription in a TA-independent manner. These findings advance our understanding of transcriptional regulation in RCNMV and provide novel insights into the origin of the TA–TABS interaction.
Highlights
Members of the positive-sense RNA virus family Tombusviridae [1] express certain encoded proteins via the production of subgenomic mRNAs [2,3,4,5,6,7,8]
Secondary structure models for the TA and trans-activator binding site (TABS), (ii) revealed that the TABS is partially base paired with proximal upstream sequences, which limits TA access, (iii) demonstrated that the aforementioned intra-RNA1 base pairing involving the TABS modulates the TA–TABS interaction in vitro and sg mRNA levels during infections, and (iv) revealed that the TABS in RNA1 can be modified to mediate sg mRNA transcription in a TA-independent manner
Attenuation RNA structures are formed by localized genomic sequences [3,6], long-distance RNA–RNA interactions [11,12], or RNA–RNA interactions between genome segments [13], and may adopt simple RNA secondary structures [3,6] or elaborate higher-order RNA conformations created by genome-level folding [12]
Summary
Members of the positive-sense RNA virus family Tombusviridae [1] express certain encoded proteins via the production of subgenomic (sg) mRNAs [2,3,4,5,6,7,8]. Viruses 2021, 13, 2252 is the third ORF encoded in RNA1 and is expressed via transcription of a sg mRNA (1.5 kb) during infections (Figure 1B) [13]. The location of the transcription initiation site of the sg mRNA in RNA1 is indicated by indicated by the angled arrow. Relative reactivity of nucleotides are depicted as red, green, complementary to the TABS. The TABS sequence is depicted by green shading and the and the TP-SL structure is shown in the dotted grey box. Analysis of the RNA1 TABS and TP-SL structures in RCNMV, SCNMV, and CRSV
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