RESUMEN
Species A rotavirus non-structural protein 3 (NSP3) is a translational regulator that inhibits or, under some conditions, enhances host cell translation. NSP3 binds to the translation initiation factor eIF4G1 and evicts poly-(A) binding protein (PABP) from eIF4G1, thus inhibiting translation of polyadenylated mRNAs, presumably by disrupting the effect of PABP bound to their 3'-ends. NSP3 has a long coiled-coil region involved in dimerization that includes a chaperone Hsp90-binding domain (HS90BD). We aimed to study the role in NSP3 dimerization of a segment of the coiled-coil region adjoining the HS90BD. We used a vaccinia virus system to express NSP3 with point mutations in conserved amino acids in the coiled-coil region and determined the effects of these mutations on translation by metabolic labeling of proteins as well as on accumulation of stable NSP3 dimers by non-dissociating Western blot, a method that separates stable NSP3 dimers from the monomer/dimerization intermediate forms of the protein. Four of five mutations reduced the total yield of NSP3 and the formation of stable dimers (W170A, K171E, R173E and R187E:K191E), whereas one mutation had the opposite effects (Y192A). Treatment with the proteasome inhibitor MG132 revealed that stable NSP3 dimers and monomers/dimerization intermediates are susceptible to proteasome degradation. Surprisingly, mutants severely impaired in the formation of stable dimers were still able to inhibit host cell translation, suggesting that NSP3 dimerization intermediates are functional. Our results demonstrate that rotavirus NSP3 acquires its function prior to stable dimer formation and remain as a proteasome target throughout dimerization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biosíntesis de Proteínas/genética , Multimerización de Proteína/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Chlorocebus aethiops , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Mutación Puntual/genética , Proteínas de Unión a Poli(A)/genética , Unión Proteica/genética , ARN Mensajero/genética , ARN Viral/genética , Rotavirus/genética , Infecciones por Rotavirus/virología , Alineación de Secuencia , Replicación Viral/genéticaRESUMEN
We describe the clinical characteristics and outcomes of adults hospitalized with pneumonia during the pandemic (H1N1) 2009 outbreak. Patients admitted to a general hospital in San Luis Potosí, Mexico, from April 10 through May 11, 2009, suspected to have influenza virus-associated pneumonia were evaluated. We identified 50 patients with suspected influenza pneumonia; the presence of influenza virus was confirmed in 18: 11 with pandemic (H1N1) 2009 virus, 5 with unsubtypeable influenza A virus, 1 with seasonal influenza A virus (H3N2), and 1 in whom assay results for seasonal and pandemic (H1N1) 2009 viruses were positive. Eighteen patients were treated in the intensive care unit, and 10 died. During the pandemic (H1N1) 2009 outbreak, severe pneumonia developed in young adults who had no identifiable risk factors; early diagnosis and treatment of influenza virus infections may have a determinant role in outcome.