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Updated: Apr 12, 2026

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants
Published on: June 9, 2022
Rapid evolution of in vivo-selected sequences and structures replacing 20% of a subviral RNA
Allison M Murawski1, Johnathan L Nieves1, Maitreyi Chattopadhyay2
1Department of Biology, Dickinson College, Carlisle, PA 17013, USA.
Abstract:
The 356 nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) is composed of 5' sequences from a second TCV satRNA (satD) and 3' sequences derived from TCV. SHAPE structure mapping revealed that 76 nt in the poorly-characterized satD-derived region form an extended hairpin (H2). Pools of satC in which H2 was replaced with 76, 38, or 19 random nt were co-inoculated with TCV helper virus onto plants and satC fitness assessed using in vivo functional selection (SELEX). The most functional progeny satCs, including one as fit as wild-type, contained a 38-39 nt H2 region that adopted a hairpin structure and exhibited an increased ratio of dimeric to monomeric molecules. Some progeny of satC with H2 deleted featured a duplication of 38 nt, partially rebuilding the deletion. Therefore, H2 can be replaced by a 38-39 nt hairpin, sufficient for overall structural stability of the 5' end of satC.
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