Identificación y caracterización de un motivo de RNA similar al elemento GAIT en el extremo 3' del genoma del TGEV que modula la respuesta inmune innata
Author
Márquez Jurado, SilviaEntity
UAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)Date
2015-04-30Subjects
Reacción inmunitaria - Tesis doctoral; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-04-2015Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
Coronaviruses (CoVs) are enveloped, positive-sense RNA viruses that belong to
the order Nidovirales, causing respiratory and enteric infections in a wide range of
animals and human. CoV replication and transcription take place at cytoplasmic
double membrane vesicles and is mediated by the viral replicase. These processes
require the specific recognition of RNA cis-acting signals located at the ends of the
viral genome. Similarly to many other RNA viruses, the virus-encoded replication
complex presumably associates with host-cell proteins to complete the synthesis of
viral RNA. Using transmissible gastroenteritis coronavirus (TGEV) as a model, we
previously identified nine cellular proteins interacting with the genome 3’ end,
including the heterogeneous nuclear ribonucleoproteins (hnRNPs) A0, A1, A2/B1, Q
and U, the translational factors glutamyl-prolyl-tRNA synthetase (EPRS), arginyl-tRNA
synthetase (RRS) and poly(A)-binding protein (PABP), and the p100 transcriptional
coactivator. From these proteins, a functional role on viral RNA synthesis was
reported for hnRNP Q, EPRS and PABP. In this thesis, the functional study was
extended to the proteins RRS and p100, and a positive role of both cell proteins in
the viral RNA synthesis was demonstrated by silencing analysis.
The RNA domains interacting with the cell proteins involved in TGEV RNA
synthesis were further analyzed to study their mechanisms of action. After several
RNA mapping stages, a 32-nt RNA motif located at the 3’ end of the TGEV genome
was found to specifically interact with EPRS and RRS. This interaction was also
observed during the infection, where both tRNA synthetases specifically interacted
with the viral genomic and subgenomic RNAs. Interestingly, both aminoacil tRNA
synthetases were incorporated into the viral particle, possibly through their
interaction with the viral genome RNA. This RNA motif presented high homology in
sequence and secondary structure with the gamma interferon activated inhibitor of
translation (GAIT) element, which is present at the 3’ end of several mRNAs coding
proinflammatory proteins. The GAIT element is involved in the translation silencing
of these mRNAs through its interaction with the GAIT complex [EPRS, hnRNP Q, L13a,
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] to favor the resolution of
inflammation. Similarly to the cellular GAIT element, the viral RNA domain was able
to bind the GAIT complex and inhibit the in vitro translation of a chimeric mRNA
containing this motif, suggesting that the viral RNA domain could constitute the first
GAIT-like motif described in a positive RNA virus.
To test the functional role of the GAIT-like motif in TGEV infection, two
recombinant viruses harboring mutations in this motif were engineered and
characterized. Abrogation of the GAIT-like motif did not affect virus growth in cell
cultures. However, an exacerbated innate immune response, mediated by the
melanoma differentiation-associated gene 5 (MDA5) pathway, was observed in cells
infected with the mutant viruses compared with the parental virus infection.
Furthermore, mutant viruses were more sensitive to interferon beta than the
parental virus. Altogether, these data suggested that the GAIT-like motif modulates
the host innate immune response.
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