Relevancia del motivo de unión PDZ de la proteína E del coronavirus causante del síndrome respiratorio agudo y grave en patogénesis
Entity
UAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)Date
2015-06-15Subjects
Infecciones por coronavirus - Tesis doctorales; Aparato respiratorio - Enfermedades - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-06-2015Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
Severe
acute
respiratory
syndrome
coronavirus
(SARS-‐CoV)
is
the
etiological
agent
of
a
worldwide
epidemic
infecting
8000
people
with
a
mortality
of
about
10%.
A
recombinant
SARS-‐CoV
lacking
the
multifunctional
envelope
(E)
protein
generated
in
our
laboratory
was
attenuated
in
vivo,
being
a
promising
vaccine
candidate.
In
this
thesis,
we
have
combined
multidisciplinary
approaches
to
characterize
E
protein
as
a
virulence
factor.
Here
we
report
that
E
protein
contains
a
PDZ-‐binding
motif
(PBM),
a
domain
potentially
involved
in
the
interaction
with
more
than
400
cellular
proteins,
is
a
major
determinant
of
SARS-‐CoV
virulence.
Removal
of
SARS-‐CoV
E
protein
PBM
using
reverse
genetics,
caused
a
reduction
in
the
deleterious
exacerbation
of
the
immune
response
triggered
during
infection
with
the
parental
virus,
drastically
diminishing
lung
damage
and
leading
to
virus
attenuation.
Cellular
protein
syntenin
was
identified
to
bind
the
E
protein
PBM
during
SARS-‐
CoV
infection
by
using
three
complementary
strategies,
yeast
two-‐hybrid
platform,
reciprocal
coimmunoprecipitation
and
confocal
microscopy
assays.
In
addition,
syntenin
redistributed
from
the
nucleus
to
the
cell
cytoplasm
colocalizing
with
E
protein
during
infection
with
viruses
containing
the
E
protein
PBM,
activating
p38
MAPK
and
leading
to
the
overexpression
of
inflammatory
cytokines.
In
fact,
silencing
of
syntenin
using
siRNAs
led
to
a
decrease
in
p38
MAPK
activation
in
SARS-‐CoV
infected
cells,
further
reinforcing
their
functional
relationship.
Furthermore,
active
p38
MAPK
was
reduced
in
the
lungs
of
mice
infected
with
SARS-‐CoVs
lacking
E
protein
PBM
as
compared
with
those
infected
with
the
parental
virus,
leading
to
a
decreased
expression
of
inflammatory
cytokines
and
to
virus
attenuation.
Interestingly,
administration
of
a
p38
MAPK
inhibitor
increased
mice
survival
up
to
80%
after
infection
with
SARS-‐CoV.
The
relevance
of
the
E
protein
PBM
in
SARS-‐CoV
infection
was
reinforced
by
showing
that
recombinant
viruses
lacking
the
E
protein
(SARS-‐CoV-‐ΔE)
incorporated,
after
serial
passages,
novel
chimeric
proteins
containing
a
PBM
at
the
end
of
the
carboxy-‐terminal
domain.
Overall,
the
results
indicated
that
the
PBM
of
E
protein
is
a
virulence
factor
that
increases
virus
fitness
and
activates
immunopathology
preferentially
by
using
syntenin
as
a
mediator
of
p38
MAPK
induced
inflammation
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Texto de la Tesis Doctoral
Google Scholar:Jiménez Guardeño, José Manuel
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