Translation-Independent Roles of RNA Secondary Structures within the Replication Protein Coding Region of Turnip Crinkle Virus

Abstract

RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.

The AC2 Protein of a Bipartite Geminivirus Stimulates the Transcription of the BV1 Gene Through Abscisic Acid Responsive Promoter Elements

“…Constructs. The Core35S-GFP construct was based on pAI101, a binary vector modified from pCambia1300 [28,29]. An expression cassette consisting of the core 35S promoter [the last 99 nt of the cauliflower mosaic virus (CaMV) 35S promoter], the EGFP cDNA, and the poly-A signal of CaMV 35S RNA, was inserted into pAI101 at the PstI site to create Core35S-GFP.…”

“…The corresponding DNA fragments were custom-synthesized by Integrated DNA Technologies (IDT, Coralville, IA) as gBlocks fragments, and used to replace the Core35S sequence via Gibson Assembly cloning (New England Biolabs, Ipswich, MA). The AC2-expressing construct was also made in pAI101, in which the AC2 cDNA was preceded by the CaMV 35S promoter with double enhancers (2X35S), plus a translational enhancer derived from tobacco etch virus (TEV TE) [28][29][30]. The identity of all constructs was verified with Sanger sequencing.…”

“…Upon verification, the constructs were introduced into Agrobacterium tumefaciens strain C58C1 with electroporation [30]. In most experiments, various combinations of Agrobacterium suspensions were mixed together and delivered into Nicotiana benthamiana leaves as described [28][29][30][31][32]. A p19-expressing Agrobacterium strain was included in all combinations to alleviate RNA silencing-mediated mRNA degradation.…”

The AC2 Protein of a Bipartite Geminivirus Stimulates the Transcription of the BV1 Gene through Abscisic Acid Responsive Promoter Elements

“…The Core35S-GFP construct was based on pAI101, a binary vector modified from pCambia1300 [28,29]. An expression cassette consisting of the core 35S promoter (the last 99 nt of the cauliflower mosaic virus (CaMV) 35S promoter), the EGFP cDNA, and the poly-A signal of CaMV 35S RNA was inserted into pAI101 at the PstI site to create Core35S-GFP.…”

“…The corresponding DNA fragments were custom-synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA) as gBlocks fragments and used to replace the Core35S sequence via Gibson Assembly cloning (New England Biolabs, Ipswich, MA, USA). The AC2-expressing construct was also made in pAI101, in which the AC2 cDNA was preceded by the CaMV 35S promoter with double enhancers (2X35S), plus a translational enhancer derived from the tobacco etch virus (TEV TE) [28][29][30]. The identity of all constructs was verified with Sanger sequencing.…”