Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells

Abstract

BackgroundRotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types.MethodsTransport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab)2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls.ResultsOnly full-length (FL), endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells.ConclusionsThe intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study.

Rotavirus NSP4 is secreted from infected cells as an oligomeric lipoprotein and binds to glycosaminoglycans on the surface of non-infected cells

“…We reported apical secretion of NSP4 from rotavirus infected Caco-2 cells as an intact species without proteolytic cleavage [7]. Secretion of the unprocessed fully glycosylated form of NSP4 was surprising given the presence of a transmembrane domain but has been confirmed recently by Gibbons et al, [8]. The results presented here indicate that NSP4 is secreted as an oligomeric lipoprotein in complex with phospholipid.…”

Rotavirus non-structural proteins: structure and function

“…The ability of NSP4 to differentially interact with multiple viral and cellular proteins, including calnexin [68], laminin-3, fibronectin [69], caveolin [56], integrin I domains [**55], and tubulin [60] (Figure 3A) may be attributed to localization of NSP4 to different cellular compartments (ER [70], the ER-Golgi intermediate compartment (ERGIC), LC3-positive autophagosomes surrounding viroplasms [71], microtubules [60,63], membrane rafts [72,73], the exofacial surface of the plasma membrane [74], and the extracellular matrix [**55,69]) during different stages of infection and possibly to distinct forms or conformations of this multifunctional protein.…”

Novel Pentameric Structure of the Diarrhea-Inducing Region of the Rotavirus Enterotoxigenic Protein NSP4

“…It is also known to interact with other rotaviral proteins (2,23,33,37) and cellular proteins, such as tubulin, caveolin, laminin-␤3, fibronectin, and integrin (7,38,43,45,50). Besides its association with several cellular compartments, it is also secreted in both cleaved (51) and native (10,20) forms from infected and NSP4 gene-transfected cells, which was proposed previously to bind to receptors on neighboring cells and induce diarrhea (43).…”

“…During different stages of rotaviral infection, NSP4 has been proposed to be associated with diverse functions, such as the viroporin-associated alteration of Ca 2ϩ homeostasis by the release of Ca 2ϩ from the ER (16,24,47), the modification of membrane permeability (39), the triggering of an extracellular Ca 2ϩ signaling pathway (15,16), and interactions with and the transport of double-layered particles (DLPs) into the lumen of the ER for maturation into triple-layered particles (TLPs) (2,37). Although it was originally proposed to be an ER-anchored intracellular receptor for DLPs (2,5,11,37,46), several studies showed its presence in several other cellular sites, including the ER-Golgi intermediate compartment (ERGIC) (6,50), LC3-positive autophagosomes, and a novel membrane compartment colocalizing with viroplasms (6), membrane rafts (45), microtubules (50), the exofacial surface of the plasma membrane (20), and the extracellular matrix (7). While the N-terminal hydrophobic region anchors the protein to the ER or plasma membrane, the C-terminal part of the protein (residues 45 to 175) is oriented toward the cytoplasm or the extracellular environment (5,11,18,20).…”

“…Although it was originally proposed to be an ER-anchored intracellular receptor for DLPs (2,5,11,37,46), several studies showed its presence in several other cellular sites, including the ER-Golgi intermediate compartment (ERGIC) (6,50), LC3-positive autophagosomes, and a novel membrane compartment colocalizing with viroplasms (6), membrane rafts (45), microtubules (50), the exofacial surface of the plasma membrane (20), and the extracellular matrix (7). While the N-terminal hydrophobic region anchors the protein to the ER or plasma membrane, the C-terminal part of the protein (residues 45 to 175) is oriented toward the cytoplasm or the extracellular environment (5,11,18,20). It is also known to interact with other rotaviral proteins (2,23,33,37) and cellular proteins, such as tubulin, caveolin, laminin-␤3, fibronectin, and integrin (7,38,43,45,50).…”