RESUMEN
We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28gamma, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways.
Asunto(s)
Autoantígenos/metabolismo , Hepacivirus/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Proteínas del Núcleo Viral/metabolismo , Línea Celular , Humanos , Lisina/química , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Procesamiento Proteico-Postraduccional , UbiquitinaciónRESUMEN
We present 2 cases with severe necrotizing pneumonia due to community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection. The patients were a 89-year-old man and a male student of 15 years of age. Chest X-rays and CT scans demonstrated multiple consolidations with cavitary lesions showing necrotizing pneumonia. MRSA strains were isolated from the sputum cultures on admission in these patients who did not have any established risk factors for MRSA infections such as history of hospitalization, surgery, hemodialysis, the presence of a permanent indwelling catheter or percutaneous medical device, and residence in a long-term care facility. These patients thus satisfied the international criteria for CA-MRSA presented by the Centers for Disease Control and Prevention (CDC). Unfortunately, the first case died of CA-MRSA pneumonia in spite of intensive treatments including anti-MRSA antibiotics. Unlike the severe CA-MRSA cases in western countries, Panton-Valentine leukocidin (PVL) genes were not detected in the present cases, suggesting that factors other than PVL had a significant effect on the severity of necrotizing pneumonia. To the best of our knowledge, this is the first report of severe necrotizing pneumonia caused by CA-MRSA in Japan.
Asunto(s)
Neumonía Estafilocócica/microbiología , Staphylococcus aureus/aislamiento & purificación , Adolescente , Anciano de 80 o más Años , Antibacterianos/administración & dosificación , Toxinas Bacterianas/genética , Infecciones Comunitarias Adquiridas , Quimioterapia Combinada , Exotoxinas/genética , Resultado Fatal , Humanos , Inmunoglobulinas/administración & dosificación , Inmunoglobulinas Intravenosas/administración & dosificación , Leucocidinas/genética , Masculino , Resistencia a la Meticilina , Necrosis , Neumonía Estafilocócica/diagnóstico , Neumonía Estafilocócica/patología , Neumonía Estafilocócica/terapia , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.