The role of lipid rafts in the early stage of Enterovirus 71 infection.

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.

Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway.

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.

Application of HBx-induced anti-URGs as early warning biomarker of cirrhosis and HCC.

BACKGROUND: Hepatitis B virus (HBV) carriers are at high risk for the development of hepatocellular carcinoma (HCC), but there are no reliable markers that will identify such high-risk patients. HBV up-regulates the expression of selected genes (URGs) in the liver during chronic infection. These aberrantly expressed proteins trigger corresponding antibodies (anti-URGs) that appear prior to the detection of HCC. This study was undertaken to see if the anti-URGs could be used as early warning biomarker of HBV-induced liver cirrhosis and HCC. METHODS: A cross sectional study using a total of 625 serum samples from HBV infected and uninfected controls were tested for the anti-URGs using specific ELISAs. RESULTS: The number and specificity of anti-URGs correlated with the severity of liver disease Anti-URGs were predominantly present among patients with HBV-associated HCC (55.2%) and cirrhosis (60.7%), and at a lower frequency among patients with chronic hepatitis (35.8%), and at still lower frequencies in most asymptomatic carriers (12.3%) with normal ALT, among patients with chronic hepatitis C (38.5%) and blood donors (0.9%). These anti-URGs were rarely detected in sera from those with tumors other than HCC, except among HBV infected patients with cholangioicarcinoma and in some patients with drug induced hepatitis. 3 or more anti-URGs could precede the diagnosis of cirrhosis or HCC 11.8 months on average, and HBV hepatitis patients with 3 or more anti-URGs have much higher risk (5/20 vs 0/30) to develop cirrhosis and HCC than those patients with less anti-URGs. As the early warning biomarker, 3 or more anti-URGs were served as the threshold to separate the cirrhosis and HCC from others with a moderate sensitivity (58.3%) and specificity (80.0%), which was better than other biomarkers (AFP, AFP-L3, GPC3 and GP73) and would improve up to 70.3% when combined with another biomarker. CONCLUSIONS: The results of this clinical validation study suggest that the anti-URGs might have diagnostic/prognostic utility among patients at high risk for the development of cirrhosis and HCC.