Rotavirus Spike Protein VP4 Is Present at the Plasma Membrane and Is Associated with Microtubules in Infected Cells

Abstract

VP4 is an unglycosylated protein of the outer layer of the capsid of rotavirus. It forms spikes that project from the outer layer of mature virions, which is mainly constituted by glycoprotein VP7. VP4 has been implicated in several important functions, such as cell attachment, penetration, hemagglutination, neutralization, virulence, and host range. Previous studies indicated that VP4 is located in the space between the periphery of the viroplasm and the outside of the endoplasmic reticulum in rotavirus-infected cells. Confocal microscopy of infected MA104 monolayers, immunostained with specific monoclonal antibodies, revealed that a significant fraction of VP4 was present at the plasma membrane early after infection. Another fraction of VP4 is cytoplasmic and colocalizes with ␤-tubulin. Flow cytometry analysis confirmed that at the early stage of viral infection, VP4 was present on the plasma membrane and that its N-terminal region, the VP8* subunit, was accessible to antibodies. Biotin labeling of the infected cell surface monolayer with a cell-impermeable reagent allowed the identification of the noncleaved form of VP4 that was associated with the glycoprotein VP7. The localization of VP4 was not modified in cells transfected with a plasmid allowing the expression of a fusion protein consisting of VP4 and the green fluorescent protein. The present data suggest that VP4 reaches the plasma membrane through the microtubule network and that other viral proteins are dispensable for its targeting and transport.Rotaviruses are the most important etiologic agents of severe dehydrating infantile gastroenteritis in developed and developing countries (17). They are responsible for more than 850,000 deaths per year (14). As a member of the Reoviridae family, rotavirus has a segmented double-stranded RNA genome, enclosed in a viral capsid constituted of three concentric protein layers (37). Electron microscopy studies show that viral morphogenesis begins in cytoplasmic inclusions, termed viroplasms, where the central core and single-shelled particles are assembled (3, 10). VP4 is an unglycosylated protein and forms spikes that project from the outer layer of mature virions, which is mainly constituted by the glycoprotein VP7 (1, 34). VP4 has been implicated in several important functions, such as cell attachment, penetration, hemagglutination, neutralization, virulence, and host range (5,12,18,23). It has been shown that the infectivity of rotaviruses is increased and is probably dependent on trypsin treatment of the virus (11). This proteolytic treatment results in the specific cleavage of VP4 to polypeptides VP8* and VP5*, which represent, respectively, the amino-and carboxyl-terminal regions of the protein (22). VP4 possesses a conserved hydrophobic region located between amino acids 384 and 401 that shares some homology with the internal fusion sites of Semliki Forest virus and Sindbis virus E1 spike proteins (25). Recently, it has been shown that VP5*, which includes this hydrophobic domain, is a specific membrane-p...

Silencing the Morphogenesis of Rotavirus

“…3). The mixed pattern of VP4 that has been previously reported (15,24), which consists of perinuclear ring-like or semicircular structures closely associated to viroplasms (15) and a filamentous array due to its association with microtubules (24), was observed in cells transfected with the control siRNA and siRNA VP7 ; however, in the presence of siRNA NSP4 , most of the perinuclear structures disappeared while the filamentous signal of VP4 remained and was even more evident than that of control cells (Fig. 3).…”

Spike Protein VP4 Assembly with Maturing Rotavirus Requires a Postendoplasmic Reticulum Event in Polarized Caco-2 Cells

“…However, neither the targeting of VP4 to the cell surface nor the association of VP4 with rafts was modified by the drug, demonstrating that the fate of VP4 was independent of the accumulation of immature viral particles within the ER. The presence of VP4 on the cell surface of MA 104 cells (26) and at the apical pole of Caco-2 cells (32) has already been observed. In this study, analysis by flow cytometry clearly showed that TM did not prevent VP4 trafficking to the cell surface.…”

Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

“…These observations include electron cryomicroscopy reconstructions showing a VP4 domain buried beneath the VP7 layer (58), immunoelectron microscopy localizing VP4 in the cytoplasm between viroplasms and the ER membrane (46), colocalization of VP4 with NSP4 by immunofluorescence microscopy and biochemical assays (26), the detection of VP4 in transiently enveloped particles (48), and the accumulation of viral particles in the ER of infected cells depleted of VP4 by small interfering RNA (11). On the other hand, immunofluorescence microscopy using monoclonal antibody 7.7 detects VP4 in lipid rafts, bound to cytoskeletal elements, and exposed on the external face of the apical plasma membrane (14,38,52). This antibody does not detect VP4 in the ER lumen.…”