It remains thus possible that inhibition of efficient assembly of these signaling proteins or inhibition of activation of any of these factors could be contributing to the block in uptake. Cholesterol-rich membrane domains have been shown to mediate entry of enveloped flaviviruses such as dengue virus and Japanese encephalitis virus. In addition, filipin was shown to block HIV gag protein entry and translocation to MVBs in a clathrin-independent pathway. We found here that choleTulathromycin B sterol domains were also needed for the biogenesis of cytoplasmic EV1-induced multivesicular bodies. Ketoconazole treatment, which caused accumulation of lanosterol and lowered the amount of cholesterol in cells, caused integrin depletion out from detergent-resistant domains and had a drastic effect on internalization. The endosomal structures that were observed in the cytoplasm were early endosomal type tubulovesicular structures, suggesting that sufficient content of cholesterol in the membranes is needed for the formation of multivesicularity. In line with these results, the deletion of yeast OSH genes encoding oxysterol-binding proteins alters intracellular sterol distribution and causes vacuolar fragmentation. On the other hand, overexpression of one sterol lipid carrier ORP1L was shown to cause enlarged multivesicular bodies with higher amount of internal membranes altogether suggesting that sterol abundance and distribution is important for the biogenesis and function of endosomes. We showed further that cholesterol-rich domains are cointernalized and exist in the formed a2-MVBs. This was proven by cholesterol labeling with filipin and by the classical cold Triton X-100 treatment, which had no solubilizing effect on the a2MVBs whereas it had a more pronounced effect in the control endosomal structures labeled and induced by clustered aV integrin. This was expected as we had recently shown that cholesterol binding caveolin-1 rich domains are also accumulating in a2-MVBs. We also knew from our previous studies that clustered aV integrin is internalized via clathrin coated pits and Folinic acid calcium salt pentahydrate colocalizing with EEA1 and transferrin unlike EV1. Although it has been shown that different endosomes contain lipid raft components, our results suggest that a2-MVBs, originated from the cholesterol-rich plasma membrane domains contain a higher amount of detergent-insoluble lipids than the antibody clustered aV integrin structures studied here. Experiments with filipin and nystatin clearly showed that EV1 inside a2-MVBs was sensitive to these drugs. Administration of these drugs halted virus infection at the a2-MVBs. While drug addition during the active replication period was not much affected, administration during the early time points totally prevented infection. The later replication phase has been shown to be sensitive for cholesterol perturbation for hepatitis C virus, West Nile virus and Japanese encephalitis virus. In the case of West Nile virus, virus causes redistribution of cholesterol from the plasma membrane and recruits cholesterol to the dsRNA-positive replication sites. Hepatitis C virus RNA was shown to accumulate in detergent-insoluble vesicles and to colocalize with caveolin-2. Also flaviviral non-structural proteins were shown to associate with detergent-resistant membranes. In contrast, labeling of dsRNA during EV1 replication showed that indeed, dsRNA-positive replication sites did not accumulate cholesterol, further suggesting that the replication step of EV1 is not particularly dependent on membranes enriched in cholesterol.