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

Abstract

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...

Hsp70 Negatively Controls Rotavirus Protein Bioavailability in Caco-2 Cells Infected by the Rotavirus RF Strain

“…In these cells, both rotavirus and Hsp70 are released before any detectable cell lysis, through an atypical pathway involving lipid rafts (3,21,35). In the present work, we found that the Hsp70 level rapidly increases in response to rotavirus infection in Caco-2 cells and that this virus stress induction is specific to the molecular chaperone Hsp70.…”

Virus Infection and Lipid Rafts

“…50,51) A rotavirus follows an atypical pathway to the cell membrane by the way of the Golgi apparatus. A direct interaction of VP4, which is the most peripheral protein of the triple-layered structure of a rotavirus, with lipid rafts promotes assembly and atypical targeting of the rotavirus in intestinal cells.…”

Role of Membrane Rafts in Viral Infection

“…The role of membrane rafts in the intracellular assembly of nonenveloped viruses has been reported only for rotavirus [29,[138][139][140][141] and bluetongue virus [142], belonging to the family Reoviridae. Rotavirus replication occurs in large inclusions (known as viroplasm) in the cell cytoplasm, where viral RNA is replicated and double-layered particles are assembled.…”

“…In this process, the involvement of transient enveloped particles with rafts is highly questionable since rafts are thought to be absent from the ER. Association of VP4 with rafts in the extrareticular compartment promotes rotavirus morphogenesis as a final assembly platform and apical targeting toward the release process [29,138,139], which are significantly dependent on the raft heterogeneity (the nature and proportion of phospholipids and glycosphingolipids) [140]. Moreover, NSP4 undergoes Golgi networkspecific processing for its N-glycans through caveola-dependent Golgi network-bypassing transport [141].…”

“…The role of membrane rafts in entry of nonenveloped viruses has been investigated for simian virus 40 (SV40; Papovaviridae) [4][5][6][7][8][9][10][11][12], BK virus (Papovaviridae) [13][14][15], JC virus (Papovaviridae) [16], bovine papillomavirus (Papovaviridae) [17], human papillomavirus (HPV; Papovaviridae) [18][19][20][21][22][23][24][25][26], rotavirus (Reoviridae) [27][28][29], echovirus type 1 [30] and 11 (Picornaviridae) [31][32][33][34][35], enterovirus (Picornaviridae) [31], rhinovirus (Picornaviridae) [36], Coxsackievirus A9 and B4 (CAV; Picornaviridae) [37][38][39], and species C human adenovirus (HAdV; Adenoviridae) [40,41].…”