Infectious Clones of Tomato Chlorosis Virus: Toward Increasing Efficiency by Introducing the Hepatitis Delta Virus Ribozyme

Tomato chlorosis virus (ToCV) is an emergent plant pathogen that causes a yellow leaf disorder in tomato and other solanaceous crops. ToCV is a positive-sense, single stranded (ss)RNA bipartite virus with long and flexuous virions belonging to the genus Crininivirus (family Closteroviridae). ToCV is phloem-limited, transmissible by whiteflies, and causes symptoms of interveinal chlorosis, bronzing, and necrosis in the lower leaves of tomato accompanied by a decline in vigor and reduction in fruit yield. The availability of infectious virus clones is a valuable tool for reverse genetic studies that has been long been hampered in the case of closterovirids due to their genome size and complexity. Here, attempts were made to improve the infectivity of the available agroinfectious cDNA ToCV clones (isolate AT80/99-IC from Spain) by adding the hepatitis delta virus (HDV) ribozyme fused to the 3′ end of both genome components, RNA1 and RNA2. The inclusion of the ribozyme generated a viral progeny with RNA1 3′ ends more similar to that present in the clone used for agroinoculation. Nevertheless, the obtained clones were not able to infect tomato plants by direct agroinoculation, like the original clones. However, the infectivity of the clones carrying the HDV ribozyme in Nicotiana benthamiana plants increased, on average, by two-fold compared with the previously available clones.

Inducible Bcl-2 gene RNA interference mediated by aptamer-integrated HDV ribozyme switch

We designed HDV ribozymes with an embedded theophylline aptamer and pri-miRNA of endogenous gene Bcl-2 to engineer an RNAi-regulatory device.

Regulation of MAP4K4 gene expression by RNA interference through an engineered theophylline-dependent hepatitis delta virus ribozyme switch

We chose the HDV ribozyme with an embedded theophylline aptamer as the sensor domain and the pri-miRNA as the effector domain to engineer a gene-regulatory device, and developed a new approach for inducible RNAi in the control of MAP4K4 gene expression.

The role of an active site Mg2+ in HDV ribozyme self-cleavage: insights from QM/MM calculations

The specific position and coordination of active site Mg2+ ion have a significant direct impact on the self-cleavage reaction in HDV ribozyme.

Application of a fluorescently labeled trans-acting antigenomic HDV ribozyme to monitor antibiotic–RNA interactions

In order to determine if a fluorescently labeled trans-acting antigenomic HDV ribozyme can be used to monitor antibiotic–RNA interactions, several parameters that can influence assay sensitivity and reproducibility were tested and optimized.