Relevancia del motivo de unión PDZ de la proteína E del coronavirus causante del síndrome respiratorio agudo y grave en patogénesis

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