Factores determinantes del reconocimiento de nucleótidos en el virus de la fiebre aftosa
Advisor
Domingo Solans, EstebanEntity
UAM. Departamento de Biología Molecular; Centro de Biología Molecular Severo Ochoa (CBM)Date
2014-04-11Subjects
Aftovirus - Tesis doctorales; Fiebre aftosa - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita, leída en Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 11/04/2014Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
RNA virus populations consist of mutant spectra termed quasispecies. The
large heterogeneity in quasispecies is influenced by high mutation rates, due to the low
fidelity of the viral polymerase (RNA dependent RNA polymerase or RdRP). The
RdRPs polymerize with copy fidelity close to a theoretical error threshold, above which
there is a loss of genetic information. Infringement of this threshold by increasing the
error rate is known as entry into error catastrophe and can be driven by the use of
mutagenic agents. This antiviral strategy is termed lethal mutagenesis. The main
objective of this PhD thesis is to characterize the viral determinants that are involved in
sensitivity of foot-and-mouth disease virus (FMDV) to mutagenic agents.
FMDV belongs to the family Picornaviridae, a group of positive single stranded
RNA viruses associated with a large number of human and animal diseases. Ribavirin
(R) and 5-fluorouracil (FU) are mutagenic for FMDV. Passaging FMDV in the presence
of R or FU resulted in the selection of escape mutants. Here, we characterize the
mutants containing the substitutions G62S and M296I in protein 3D (FMDV's
polymerase) that are involved in R resistance, as well as the mutant containing the
substitution V173I in 3D, selected upon treatment with FU. In vitro studies with purified
polymerases have shown that these 3D substitutions confer an altered capacity to use
the nucleotide analogues as substrates, as compared with the wild-type (wt) enzyme.
Substitution V173I helps to correct the unbalanced mutational bias observed in the
presence of FU, presumably by modulating nucleotide incorporation parameters. In
addition, 3Ds with substitutions in residues 18 and 20 showed a highly impaired RNA
binding and a surprising increased capacity to use ribavirin-triphosphate (RTP) as
substrate. These studies led to the identification of a fidelity determinant at position
M16, located in a key position for recognition of incoming nucleotides.
Also, we have expressed and purified a truncated form of FMDV 2C, a viral
protein also involved in sensitivity to mutagenic agents. In this PhD thesis we show that
2C helps to unwind double stranded DNA in an ATP-independent manner. This result
can help to unveil the role of 2C in viral replication and the molecular mechanisms by
which 2C confers decreased sensitivity to lethal mutagenesis.
Files in this item
Google Scholar:Higuera Hernández, Ignacio de la
This item appears in the following Collection(s)
Related items
Showing items related by title, author, creator and subject.
-
Reconocimiento del virus de la fiebre aftosa por anticuerpos: implicaciones en variabilidad antigénica
Hernández Gil, Francisco Javier
1994