RESUMO
BACKGROUND/AIMS: Although it has been widely accepted that Enterovirus 71 (EV71) enters permissive cells via receptor-mediated endocytosis, the details of entry mechanism for EV71 still need more exploration. This study aimed to investigate the role of lipid rafts in the early stage of EV71 Infection. METHODS: The effect of cholesterol depletion or addition of exogenous cholesterol was detected by immunofluorescence assays and quantitative real-time PCR. Effects of cholesterol depletion on the association of EV71 with lipid rafts were determined by flow cytometry and co-immunoprecipitation assays. Localization and internalization of EV71 and its receptor were assayed by confocal microscpoy and sucrose gradient analysis. The impact of cholesterol on the activation of phosphoinositide 3'-kinase/Akt signaling pathway during initial virus infection was analyzed by Western-blotting. RESULTS: Disruption of membrane cholesterol by a pharmacological agent resulted in a significant reduction in the infectivity of EV71. The inhibitory effect could be reversed by the addition of exogenous cholesterol. Cholesterol depletion post-infection did not affect EV71 infection. While virus bound equally to cholesterol-depleted cells, EV71 particles failed to be internalized by cholesterol-depleted cells. EV71 capsid protein co-localized with cholera toxin B, a lipid-raft-dependent internalization marker. CONCLUSION: Lipid rafts play a critical role in virus endocytosis and in the activation of PI3K/Akt signaling pathway in the early stage of EV71 infection.
Assuntos
Enterovirus Humano A/patogenicidade , Microdomínios da Membrana/metabolismo , Western Blotting , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Criança , Toxina da Cólera/metabolismo , Colesterol/metabolismo , Endocitose/efeitos dos fármacos , Enterovirus Humano A/isolamento & purificação , Enterovirus Humano A/metabolismo , Humanos , Imunoprecipitação , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , beta-Ciclodextrinas/farmacologiaRESUMO
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.
Assuntos
Dinaminas/fisiologia , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Endocitose , Concentração de Íons de Hidrogênio , Neuroblastoma/virologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Clatrina/fisiologia , Primers do DNA , Neuroblastoma/patologia , RNA Interferente Pequeno , Ratos , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
CD81, a co-receptor for hepatitis C virus (HCV), is a member of the tetraspanin superfamily and is heavily palmitoylated in the juxtamembrane cysteine residues. Palmitoylation plays an important role in protein-protein interactions and association with cholesterol-rich domains of membranes. In this study, Huh7 cells expressing wild-type or palmitoylation-defective CD81 were generated to analyze whether palmitoylation of CD81 is involved in HCV cell entry. Our data showed that de-palmitoylation of CD81 dramatically reduced its association with tetraspanin CD151, but did not influence CD81 partition in detergent-resistant membranes. Moreover, de-palmitoylated CD81 decreased the host cell susceptibility to HCV. Notably, CD151-specific antibodies and siRNA inhibited HCV cell entry, and detachment of CD81 with CD151 decreased the lateral movement of virus particle/CD81 complex to areas of cell-cell contact. These results suggest that palmitoylation of CD81 should facilitate HCV entry, at least in part, by regulating the association of CD81 with tetraspanin-enriched microdomains.