Rotaviruses are a major cause of acute gastroenteritis in infants and young children, producing a high burden of disease worldwide and over 600,000 deaths per annum, mainly in developing countries (43). Recently, two live attenuated rotavirus vaccines (49, 58) have been licensed in various countries, and their widespread use in universal mass vaccination programs is being implemented (55).Rotaviruses form a genus of the Reoviridae family. They contain a genome of 11 segments of double-stranded RNA (dsRNA) encoding six structural proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). After entry into the host cell the outer layer of the triple-layered particles (TLPs; infectious virions) is removed in endocytic vesicles, and the resulting double-layered particles (DLPs) actively transcribe mRNAs from the 11 RNA segments and release them into the cytoplasm. The mRNAs are translated into proteins but also act as templates for dsRNA synthesis (RNA replication). The early stages of viral morphogenesis and viral RNA replication occur in cytoplasmic inclusion bodies termed "viroplasms." Partially assembled DLPs are released from viroplasms and receive their outer layer in the rough endoplasmic reticulum (RER), forming TLPs (for details, see Estes and Kapikian [20]).The rotavirus nonstructural proteins NSP2 and NSP5 are major components of viroplasms (20, 47). These two proteins alone are sufficient to induce the formation of viroplasm-like structures (VLS) (21). Blocking of either NSP2 or NSP5 in rotavirus-infected cells significantly reduces viroplasm formation and the production of infectious viral progeny (11,54,57). Until now, host cell proteins involved in viroplasm formation have not been identified.Morphological similarities between viroplasms and lipid droplets (LDs) prompted us to investigate their relationship. Both structures have phosphoproteins (NSP5 and perilipin A, respectively) inserted on their surface in ringlike shapes (16,34). LDs are intracellular organelles involved in lipid and carbohydrate metabolism. They consist of a neutral lipid core surrounded by a phospholipid monolayer containing LD-associated proteins; those include proteins of the PAT family consisting of perilipin, adipophilin (adipose differentiation-related protein [ADRP]), and TIP-47 (9, 37). Lipolysis from LDs is regulated by hormones such as catecholamines, which trigger the phosphorylation of hormone-sensitive lipase (HSL) and perilipin A and induce LD fragmentation. Incubating adipocytes with the -adrenergic agonist isoproterenol and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) activates this pathway (27, 34). LD formation can also be blocked
Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol-and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.Lipids membrane microdomains are dynamic entities involved in the control of the lipid-lipid and lipid-protein interactions that play a key role in numerous cellular functions such as signal transduction and membrane transport and trafficking (27). Membrane microdomains enriched in cholesterol and sphingolipids, also termed rafts, are thought to act as transitory platforms on which lipids and proteins may interact dynamically to exert a function that may be interrupted as the microdomain dissociates. It is thought that rafts emerge from the Golgi apparatus and reach the plasma membrane through a still-discussed intracellular pathway. Among the numerous functions of microdomains so far explored, various steps of virus interactions with their host cells have been proposed (8,32,44,47,53,66,71). These findings mainly concerned enveloped viruses, whose lipid membranes are expected to interact with the host cell membranes. By contrast, nonenveloped viruses that replicate and assemble in the cytoplasm of host cells have been scarcely explored for their putative interactions with membrane microdomains (37,48).Rotavirus, a triple-layered nonenveloped virus (70), is a worldwide cause of infantile gastroenteritis, accounting for an estimated 600,000 deaths annually (2). Knowledge of the detailed process of virus assembly is required to provide a molecular basis for the design of drugs or strategies able to interfere with virus entry, assembly, and/or replication. The interest in this approach has been enhanced since the withdrawal of the tetravalent vaccine because of side effects (7). In vivo rotavirus specifically targets highly polarized intestinal cells (59). This prompted us to develop studies on rotavirus infection of Caco-2 cells (17), which originate from human colon and which display a well-polarized and differentiated enterocytic phenotype when grown...
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