Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, “naked” RNAs, that replicate through RNA-RNA transcription. Viroids of the Avsunviroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, viroids have only been detected in plant cells. We investigated the replication of Avocado sunblotch viroid (ASBVd) of the Avsunviroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrated that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We showed that the monomeric RNA of the viroid is destabilized by the nuclear 3’ and the cytoplasmic 5’ RNA degradation pathways. For the first time, our results provide evidence that viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd (Delan-Forino et al, 2011). Furthermore the characteristics of each strand of ASBVd were analyzed using biophysical analysis techniques (Delan-Forino et al, 2014). ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in their thermal denaturation profiles. Subsequently, the secondary structures of ASBVd of plus and minus polarities were probed using the RNA-selective 2’-hydroxyl acylation method and analyzed by the primer extension (SHAPE) method. The models obtained show that ASBVd of the two polarities adopt different structures. Moreover, the results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in the in vivo viroid life cycle.
In the same line of investigation we report the first Raman characterization of the structure and activity of ASBVd, for the plus and minus viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3’-endo/anti sugar pucker configuration, although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg2+ activated self-cleavage of the viroid does not induce any significant alterations of the secondary viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved viroids. Our results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes (Hui Bon Hoa et al, 2014).
Viroids of the Asunviroidae family replicate in the chloroplasts of infected hosts. It is now largely admitted that chloroplasts evolved from a cyanobacterium ancestor. Because of this phylogeny relationship, we sought if a member of the Asunviroidae could be replicated in a cyanobacterium. We demonstrate that the Avocado sunblotch viroid (ASBVd) RNA strands of (-) and (+) polarities are able to replicate in the filamentous and heterocystous cyanobacterium Nostoc PCC7120. ASBVd replication does not impair the growth or the ability of the strain to sustain cell differentiation. Our results provide the first evidence that a prokaryotic cell possesses all the machinery required to sustain RNA replication without DNA intermediate (Latifi et al, 2014 submitted). These data are of great importance in our future knowledge on the origin and the evolution of viroids.
At the interface between Biology, Physics and Chemistry, we have presently underaken studies on the stucture, functions and interactions of nucleic acids. In one approach that is well established in physics, we wish to understand the laws that direct interactions in biological systems by performing experimental studies based on model systems and by modelisation based on concepts mainly developed in physics of soft matter. The second approach is based on the concept of the structure-function relationship in biology : rather than searching for general rules, the object is to understand specificity, that is, the differences in the structure selected by evolution, by performing experimental studies in physics based on several methods such as spectroscopy, diffraction, microscopy, etc.
We are interested in the relationship, dynamics-function. At the molecular level, the activity of a biological system depends not only on the structural organisation of the system, but also on the movements that drive it. For certain activities such as catalysis, increased flexibility is required. In contrast, for others such as the transfer of electrons, a greater rigidity is essential. To explore the relationship, dynamics-function, original approaches are being introduced : a constant effective force (resilience) that can be measured by neutron diffusion, is strongly correlated with the biological function of systems. This method successfully validated on proteins of halophilic archeae demonstrates that adaptation to temperature results from forces that stabilize macromolecular structures and determine their flexibility in a specific scale of values. In collaboration with G. Zaccai at the ILL in Grenoble and A. Yonath of the Department of Structural Biology, Weizmann Institute in Rehovoth, Israel, Martina Rihova, a Ph.D. student in our laboratory is performing dynamics measurements on a ribozyme, a viroid and on a "protoribosome" under different solvent and temperature conditions. We are also starting to measure the intracellular molecular dynamics of cyanobacteria and halophilic archea in order to establish how these cells are affected by the penetration of the viroid. Developing and applying this type of methodological approach by spectrometry allowing to quantify functional intramolecular movements constitutes an important challenge that could open new areas of research. Our preliminary results suggest that during the course of evolution, selected RNAs presented specific dynamics that allowed structures and flexibility to be maintained within the narrow limits required for biological activity.