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In vivo studies on the molecular basis of the mechanism of suppression of tobacco mosaic virus (TMV) infectivity through increased mutagenesis

Dates: from 01/10/2007 to 04/10/2010
Funding: Ministerio de Educación y Ciencia
Participants: Universidad de Málaga
Principal Investigator: Ana Grande Pérez
Researchers: Ana Grande Pérez.
RNA viruses replicate with extremely high mutation rates due to the low fidelity of RNA-dependent RNA polymerases and reverse transcriptases. Mutation, recombination, short replicative cycles and high copy yields contribute to the generation of complex distributions of closely related mutant genomes able to adapt to fluctuating environments termed quasispecies. One biological implication of viral quasispecies, of great sanitary impact, consists in the emergence of resistant mutants after antiviral treatment making difficult the elimination of these viral infections. Thus, it is necessary the search for new antiviral strategies. One of such strategies is based in the elimination of RNA virus through enhanced mutagenesis using antiviral drugs that introduce mutations in the viral genome. Results with different viral systems of animal and plant RNA viruses demonstrate that certain nucleoside and base analogues are able to introduce mutations in the viral genome and eliminate infection. By means of experimental and theoretical approaches, a model of viral extinction through enhanced mutagenesis in which a fraction of the RNA molecules synthesized can behave as a defective subpopulation able to drive the viable class extinct has been proposed. Thus, two extinction pathways would exist, one at high amounts of mutagen, where the quasispecies completely loses its ability to infect and replicate, and a second one, at lower amounts of mutagen, where replication continues while the infective class gets extinct because of the action of defectors. While the majority of these studies have been performed in cultures of animal virus systems it is important to extend the results to the in vivo scenario. Plant virus systems are a convenient in vivo experimental model to study evolution of viruses subjected to chemical mutagenesis.
This project focuses on the in vivo study of the molecular bases of the suppressive mechanism of RNA virus infectivity due to enhanced mutagenesis after nucleoside and base analogue treatments. The model system chosen is the Tobacco mosaic virus (TMV) in the host Nicotiana tabacum of which we have much information not only about the virus bout also about the nucleoside and base analogue treatments that eliminate infectivity in this host. The main goals of this project are:
1. To study the TMV evolution and movement in infected plants after treatments with the base analogues 5-fluorouracilo y 2-tiouracilo and the nucleoside analogue ribavirin using antiviral established conditions.
2. To quantify TMV RNA molecules of the obtained viral populations; we will differentiate between chain polarities.
3. To analyse the genetic complexity of those RNA viral populations and determine their mutation frequencies.
4. To detect hypermutated viral RNA molecules through dHPLC (denaturing HPLC).
5. To extend the studies about the suppression of TMV through enhanced mutagenesis to Arabidopsis thaliana.
The results obtained will contribute to establish the bases of a new antiviral therapy and would see their phytosanitary application in the elimination of viruses from in vitro propagated plants with vegetative reproduction.

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