The Amino-Terminal Nine Amino Acid Sequence of Poliovirus Capsid VP4 Protein Is Sufficient To Confer N-Myristoylation and Targeting to Detergent-Insoluble Membranes

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...

Section: Discussionmentioning

confidence: 79%

mentioning

confidence: 99%

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Rafts Promote Assembly and Atypical Targeting of a Nonenveloped Virus, Rotavirus, in Caco-2 Cells

Sapin

Colard

Delmas

et al. 2002

“…77, 2003 ROLE OF POLIOVIRUS VP4 SEQUENCES DURING CELL ENTRY 5273 the kinetic differences in photosensitivity of infections with NR mutant 135S particles occur after the VP4 sequences are relocated from the particle interior and inserted into cellular membranes. Previous studies have demonstrated that the myristoyl modification and 9 residues at the VP4 N terminus are sufficient to target VP4 via intracellular trafficking pathways to detergent-insoluble membrane microdomains (19). It is possible that, during entry, VP4 sequences may be similarly targeted to detergent-insoluble membrane microdomains or lipid rafts after receptor binding on the extracellular surface of the plasma membrane.…”

Section: Discussionmentioning

confidence: 99%

Genome Delivery and Ion Channel Properties Are Altered in VP4 Mutants of Poliovirus

Danthi

Tosteson

et al. 2003

108

129

During entry into host cells, poliovirus undergoes a receptor-mediated conformational transition to form 135S particles with irreversible exposure of VP4 capsid sequences and VP1 N termini. To understand the role of VP4 during virus entry, the fate of VP4 during infection by site-specific mutants at threonine-28 of VP4 (4028T) was compared with that of the parental Mahoney type 1 virus. Three virus mutants were studied: the entry-defective, nonviable mutant 4028T.G and the viable mutants 4028T.S and 4028T.V, in which residue threonine-28 was changed to glycine, serine, and valine, respectively. We show that mutant and wild-type (WT) VP4 proteins are localized to cellular membranes after the 135S conformational transition. Both WT and viable 4028T mutant particles interact with lipid bilayers to form ion channels, whereas the entry-defective 4028T.G particles do not. In addition, the electrical properties of the channels induced by the mutant viruses are different from each other and from those of WT Mahoney and Sabin type 3 viruses. Finally, uncoating and/or cytoplasmic delivery of the viral genome is altered in the 4028T mutants: the 4028T.G lethal mutant does not release its genome into the cytoplasm, and genome delivery is slower during infection by mutant 4028T.V 135S particles than by mutant 4028T.S or WT 135S particles. The distinctive electrical characteristics of the different 4028T mutant channels indicate that VP4 sequences might form part of the channel structure. The different entry phenotypes of these VP4 mutants suggest that the ion channels may be related to VP4's role during genome uncoating and/or delivery.Poliovirus, a member of the Picornaviridae family, encapsidates its 7,400-nucleotide positive-sensed RNA genome within an icosahedrally symmetric protein shell that is formed by 60 copies of the four capsid proteins (VP1 to VP4). VP1, VP2, and VP3 form the surface of the virion, with VP1 located at each fivefold axis and VP2 and VP3 alternately positioned around each threefold axis. VP4 in its entirety as well as the amino termini of VP1, VP2, and VP3 are buried within the interior of the capsid, lying along the inner surface of the virion shell (12).Poliovirus entry into cells is initiated by binding to the poliovirus receptor (PVR) on the cell surface. PVR binding induces a conformational transition within the virus particle that leads to formation of altered particles (termed A particles) sedimenting at 135S (versus the 160S sedimentation value of the native particle) (references 8 and 23 and references therein). This conformational transition results in relocation of VP4 and the VP1 N termini from the particle interior to the virion exterior. The appearance of VP4 and VP1 domains on the particle surface is correlated with dramatic differences in the functional behavior of the native 160S and altered 135S particles. Functionally, these PVR-induced conformational rearrangements generate 135S particles that acquire the ability to bind to liposomes, to form ion channels in lipid bilayers, and ...

“…The myristate moiety could contribute to important reversible membrane interactions of the p15 N-terminal ectodomain with either donor or target membranes, analogous to the well-known "myristoyl-switch" mechanisms that facilitate reversible membrane interactions of cytosolic proteins (1,25,32,40,44). The protein-lipid interactions mediated by myristoylation also include those involved in protein targeting to specific membrane environments, such as raft microdomains at the cell periphery (14,31,42). Alternatively, myristic acid has been shown to confer localized effects on protein structure, with consequences for protein stability (29,35,59) or ligand binding (12,35,50,54).…”

Section: Discussionmentioning

confidence: 99%

Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein

Dawe

Corcoran

Clancy

et al. 2005

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusioninducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N exoplasmic /C cytoplasmic topology. This topology results in the translocation of the smallest ectodomain (ϳ20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of proteinmediated membrane fusion.The baboon reovirus (BRV) p15 protein is a novel member of the recently described fusion-associated small transmembrane (FAST) protein family (9,11,45). The FAST proteins are unusual membrane fusion proteins encoded by the fusogenic subgroup of orthoreoviruses, one of the few examples of nonenveloped viruses that induce cell-cell fusion and syncytium formation (15,18). At 10 to 15 kDa, the reovirus FAST proteins are the smallest known viral membrane fusion proteins and are unlikely to undergo the types of extensive structural rearrangements required for enveloped virus fusion protein activity (27,48). The FAST proteins are also the only examples of nonstructural viral proteins that induce membrane fusion (11, 45). As a result of their nonstructural nature, the FAST proteins play no role in reovirus entry. Their sole purpose appears to reflect enhanced dissemination of the infection via syncytium formation, following FAST protein expression in reovirus-infected cells (16,17). The unusual structural features of the FAST proteins and their unique role in the virus replication cycle suggest the mechanism of FAST-mediated membrane fusion is unlikely to adhere to the existing paradigm, which is derived from studies of the enveloped virus fusion proteins (5,48,52).In addition to BRV p15, FAST proteins have been recently characterized from avian reovirus (ARV), Nelson Bay reovirus (NBV), and reptilian reovirus (RRV) (9, 45). Although th...