Estudio de las modificaciones post-traduccionales que afectan a la proteína de la cápsida del Plum pox virus y su papel en el desarrollo del ciclo viral
Author
Hervás García, MartaEntity
UAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)Date
2019-09-23Subjects
Potyvirus - Genética - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecula. Fecha de lectura: 23-09-2019Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
O-GlcNAcylation and phosphorylation are two of the most widespread post-translational
modifications (PTMs) in eukaryotes. In animals, it has been seen that both PTMs share the same
protein targets, being common to act together in the regulation of common processes and even
affect each other (cross-talk). In plants, the best-studied target of O-GlcNAcylation is the capsid
protein (CP) of the potyvirus Plum pox virus (PPV), which is modified by SECRET AGENT (SEC) in
its N-terminal region. Previous laboratory results suggested that PPV CP, in addition to being OGlcNAcylated,
is also phosphorylated. In this work, we have demonstrated by mass
spectrometry (MS) that the CP of the Rankovic isolate of PPV (PPV-R) assembled in virions is
phosphorylated in vivo at the N-terminus (S25, S81 and S101) and the core región (S118, T254
and T313), in residues that do not coincide with O-GlcNAcylation targets. Our findings show that
PPV-R CP can be concurrently O-GlcNAcylated and phosphorylated at nearby aminoacids.
However, a differential proteomic approach based on isobaric tags for relative and absolute
quantitation (iTRAQ) showed a significant enhancement of phosphorylation at S25 in virions
recovered from O-GlcNAcylation-deficient plants (SEC-b2), suggesting that a cross-talk between
O-GlcNAcylation and phosphorylation in PPV-R CP takes place.
A combination of proteomic approaches plus protein immunodetection techniques were
applied to study the phosphorylation and O-GlcNAcylation patterns of PPV virions purified from
different hosts and corresponding to several PPV strains. We found both PTMs occur in a variety
of hosts (including the natural host Prunus persica) and modify PPV strains as different as
Dideron (D), Cherry (C) and Recombinant (Rec). However, mapping of phosphorylated residues
showed that not all the targets of this modification concur in different PPV strains. In addition,
the study of PPV mutants affected in the threonine of a CK2 motif, which is phosphorylated in
the CP of the potyvirus Potato virus A (PVA) and mostly conserved in this viral genus, revealed
that the function of phosphorylation in potyviral infection is not conserved. On the other hand,
also using immunodetection techniques and a battery of mutants and PPV subviral constructs,
we found that O-GlcNAcylation and phosphorylation of CP can take place independently of the
viral infection, but a correct folding of the protein is necessary to be O-GlcNAcylated.
While preventing PPV-R CP phosphorylation only had a limited impact on viral infection,
permanent emulation of this modification by mutating one or several of the phospho-targets to
aspartic acid affected the in vitro CP stability and its ability to form virions, as was shown by
electron microscopy and sucrose gradient sedimentation. We propose that the joint and
opposite action of O-GlcNAcylation and phosphorylation on the CP N-terminal region regulates
the protein stability, while phosphorylation in the core region controls the assembly of viral
particles.
Finally, although we have not been able to find proteins other than SEC that are involved in
the modification of PPV CP, we have generated Arabidopsis thaliana transgenic plants
expressing a functional version of a tagged SEC protein, with the aim of using them to trap SEC
interactors that could participate in the dynamic modifications of the CP.
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