Thermodynamic Properties of RNA Virus Structures
Virus-induced gene silencing (VIGS) allows for the rapid targeting of gene expression and has been instrumental for characterizing plant genes. However, foreign sequences inserted into VIGS vectors are rarely maintained for unknown reasons. Citrus yellow vein associated umbra-like virus (CY1) with its solved secondary structure was converted into a VIGS vector to determine why simple hairpins inserted into non-functional, single-stranded locations are not maintained. When CY1 contained foreign hairpins with thermodynamic properties (positional entropy and/or delta G) differing from those of natural CY1 hairpins, deletions arose within a few weeks of infecting Nicotiana benthamiana. In contrast, duplication and insertion of 4 natural CY1 hairpins (up to 200 nt) into the same locations were retained until plant senescence. Hairpins containing similar conformations and thermodynamic properties as natural hairpins were also retained, as were hairpins that shared thermodynamic properties but were conformationally distinct. By predicting and modulating these thermodynamic properties, a hairpin was retained by CY1 for at least 30 months in citrus. Other virus vectors (TNV, PVX) were found to have similar requirements for insert stability, suggesting that the properties found apply to more than just CY1. These findings strongly suggest that RNA viruses have evolved to contain substructures with specific thermodynamic properties, and hairpins containing these properties are stable when inserted into non-functional regions of the genome, opening up VIGS for long-lived trees and vines.
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We are currently working to determine additional factors that influence stability and whether large inserts can be stabilized by changing their structure to more closely resemble viral substructures
Figure legend. Design of hairpins with appropriate and inappropriate delta Gs. (A) Sequences of Ftsz160, Ftsz159, and Ftszrc160. Bold sequences are identical in Ftsz160 and Ftsz159 and are the reverse complement of the sequence in Ftszrc160. (B) Secondary structures of the three hairpins and a deletion product derived in plants from Ftsz160 (marked by yellow diamond in C). The three designed hairpins were inserted into CY1. Green arrows denote deletion end points in the unstable hairpin. (C) RT-PCR products of Ftsz1602219DH4*, Ftsz1592219DH4*, and Ftszrc1602219DH4*. (D) Graph showing relationship between delta G and hairpin length for retained and poorly retained parental, mimic, and designed hairpin inserts. Post del, hairpin formed from remaining viral sequences following deletion in the insert.