The first three-dimensional images of viroids are published, using atomic force microscopy (AFM)

The collaboration between researchers of the Center for Astrobiology (CAB, CSIC-INTA), the Institute of Materials Science of Madrid (ICMM, CSIC) and the Institute of Molecular and Cellular Biology of Plants of Valencia (IBMCP, CSIC-UPV), has allowed to obtain the first 3D images of viroids in native conditions.


Viroids are the smallest known pathogens and have so far only been found in plants, although the human hepatitis delta virus shares some properties with them. Besides the great scientific interest that they have, they are very important economically since they can produce important damages in crops such as potatoes, tomatoes, citrus fruits or ornamental plants. The infectious agent is a naked circular RNA molecule (without a capsid that protects it as it does in viruses), extremely small in size (between 246 and 434 nucleotides) and which has a unique characteristic among pathogens: it does not code for any protein. As with viruses (although following a seemingly simpler process), viroids replicate autonomously in the cells of their hosts, using their molecular machinery to generate new copies of the infectious RNA.

Since its discovery by T.O. Diener in 1971, have been described around 30 species of viroids, which are classified in the families Pospiviroidae (whose replication occurs in the nucleus of the infected cell) and Avsunviroidae (which replicate in the chloroplasts, with the participation of ' ribozymes' or RNA enzymes contained in the viroid itself). During the last decades, one of the greatest experts in viroids is Ricardo Flores, a researcher at IBMCP and co-author of this article.

Since viroid RNA does not encode proteins, its biological functions lie in the spatial conformation of RNA itself. Therefore, it is essential to know it in the most detailed way possible. Its secondary or two-dimensional structure has been previously studied using RNA folding software and using chemical modification systems in vitro and in vivo. Regarding the visualization of individual molecules of viroidal RNA, however, no progress had been made since more than 40 years ago some articles were published in which transmission electron microscopy (TEM) was used to obtain 2D images of the viroid. of the potato fusiform tuber (PSTVd, belonging to the family Pospiviroidae).

In the study published now in the journal RNA Biology, led by researchers from the Center for Astrobiology (CAB, CSIC-INTA), it has been used, as indicated by Miguel Moreno (researcher of the CAB and first signatory of the work) "a tool of nanotechnology that allows us to obtain 3D images of individual RNA molecules in native conditions and without the need to mark them or coat them with any substance, as we showed for a different biological system together with Luis Vázquez (from the ICMM) and other collaborators in a previous article: Atomic force microscopy (AFM) ". In this way, for the first time, the structure of three different viroids, PSTVd, and two species belonging to the Avsunviroidae family were able to be visualized and analyzed at nanoscale resolution: the viroid of the latent peach mosaic (PLMVd) and the latent viroid of the Eggplant (ELVd).

From each of these three viroid spices two different variants have been studied by AFM, analyzing the effect on their structure of two different ionic conditions. The bio-nanotechnological methodology used has allowed to determine the influence of the Mg2 + cation in the 3D conformation of these molecules. In addition, in the case of PLMVd, the relevance of tertiary RNA RNA interactions in the stabilization of its functional structure has been verified.

For Carlos Briones, head of the group of Molecular Evolution of the CAB and main author of the article, "in addition to being fundamental systems to study the sequence-structure-function relationships in RNA, viroids are also very interesting from the point of view of origin and the early evolution of life, since they could constitute authentic relics of the RNA World that probably existed on our planet before the appearance of cells with DNA and proteins. "




Figure: three-dimensional images of the three viroids analyzed in this study using AFM: PSTVd, PLMVd and ELVd. The bar represents 10 nanometers.

 

Fuente: UCC-CAB

 

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