phosphatidylinositol-4-phosphate and Liver-Neoplasms

West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus, which main natural hosts are birds but it also infects equines and humans, among other mammals. As in the case of other plus-stranded RNA viruses, WNV replication is associated to intracellular membrane rearrangements. Based on results obtained with a variety of viruses, different cellular processes have been shown to play important roles on these membrane rearrangements for efficient viral replication. As these processes are related to lipid metabolism, fatty acid synthesis, as well as generation of a specific lipid microenvironment enriched in phosphatidylinositol-4-phosphate (PI4P), has been associated to it in other viral models. In this study, intracellular membrane rearrangements following infection with a highly neurovirulent strain of WNV were addressed by means of electron and confocal microscopy. Infection of WNV, and specifically viral RNA replication, were dependent on fatty acid synthesis, as revealed by the inhibitory effect of cerulenin and C75, two pharmacological inhibitors of fatty acid synthase, a key enzyme of this process. However, WNV infection did not induce redistribution of PI4P lipids, and PI4P did not localize at viral replication complex. Even more, WNV multiplication was not inhibited by the use of the phosphatidylinositol-4-kinase inhibitor PIK93, while infection by the enterovirus Coxsackievirus B5 was reduced. Similar features were found when infection by other flavivirus, the Usutu virus (USUV), was analyzed. These features of WNV replication could help to design specific antiviral approaches against WNV and other related flaviviruses.

Topics: Animals; Antiviral Agents; Blotting, Western; Carcinoma, Hepatocellular; Cell Membrane; Cells, Cultured; Chlorocebus aethiops; Fatty Acids; Fluorescent Antibody Technique; Humans; Liver Neoplasms; Microscopy, Confocal; Phosphatidylinositol Phosphates; Plasmids; Real-Time Polymerase Chain Reaction; RNA, Messenger; Vero Cells; Virus Replication; West Nile Fever; West Nile virus

Tetraspanins is a large family of membrane proteins that are implicated in cell proliferation, differentiation and tumor invasion. Specifically, the tetraspanin CD81 has been involved in cell proliferation but the mechanism is unknown. Here, we show that CD81 clustering stimulates ERK/MAPKinase activity and tyrosine phosphorylation of the adapter protein Shc in Huh7 cancer cells. In addition, overexpression of CD81 in HepG2 cells, NIH3T3 cells, and murine fibroblasts GD25 lacking the beta1 family of integrins induces cell proliferation and ERK/MAPKinase activation. Linked with this event, we observed an increase in CD81-associated type II phosphatidylinositol 4-kinase activity. A mutant in the PTB domain of Shc failed to interact with phosphoinositides and localize to the plasma membrane thus blocking CD81-induced ERK/MAPKinase activation. Therefore, we conclude that CD81 stimulates synthesis of phosphoinositides with the recruitment of Shc to the plasma membrane via PTB domain, and this sequence of events induces activation of ERK/MAPKinase. These findings define a novel mechanism of ERK/MAPKinase activation and tumor cell proliferation.

Topics: 1-Phosphatidylinositol 4-Kinase; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Antigens, CD; Carcinoma; Cell Division; Cell Line, Tumor; Cytosol; Enzyme Activation; Fibroblasts; Hepatocytes; Humans; Liver Neoplasms; Membrane Proteins; Mice; Mitogen-Activated Protein Kinases; Mutation; NIH 3T3 Cells; Phosphatidylinositol Phosphates; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Tetraspanin 28