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 beta-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.

FIG. 1

Immunostaining of VP4 at the plasma membrane of infected and nonpermeabilized cell. The cell monolayer was subjected to IF as described in Materials and Methods with specific anti-VP4 MAbs. The nucleus was stained with propidium iodide. Monolayers were analyzed by confocal microscopy with optical sectioning in 1-μm increments from the cell attachment to glass to the top. (A) Gallery of images corresponding to sequential sections in 1-μm increments from bottom to top. (B) Z sectioning showing localization of VP4 (green; bottom) and the plasma membrane that was stained with WGA and conjugated with rhodamine (red; middle); colocalization appears in yellow (top). Bar = 20 μm.

FIG. 2

Localization of VP4 expressed in COS-7 cells. COS-7 cells were transfected with pEGFP-N1 (C) or pEGFP-N1-VP4 (A and B) and fixed at 48 h posttransfection, and the fluorescence was analyzed by confocal microscopy. Projection of all optical sections (A and C) and three optical sections located at the top, middle, and bottom of the transfected cell (B) are shown. Bar = 20 μm.

FIG. 3

Cell surface detection of VP4. Flow cytometry was carried out as described in Materials and Methods. (A) MA104 mock-infected cells, stained with three MAbs, RV133, 5.73, and anti-CD13, directed against VP6, VP4, and CD13, respectively. (B) Infected cells stained with anti-VP6 MAb RV133 and anti-VP4 MAb 5.73 at 6 h p.i. (C) Infected cells stained with anti-VP4 MAbs (2G4, 5.73, 1D8, 7.7, and 6.3) and an anti-VP6 MAb (RV138). (D) Cells infected with porcine rotavirus strain OSU or with bovine rotavirus strain RF and stained with MAb 5.73 against bovine VP4. Peak C in panels A to C corresponds to noninfected cell autofluorescence.

FIG. 4

Kinetics of VP4 at the surface of infected MA104 cells between 0 and 6 h p.i. Each panel shows the result at a single time p.i. and with two MAbs. Cells were stained with anti-VP6 MAb RV138, which binds to TLPs, and with MAb 5.73 directed against spike viral protein VP4. Peak C in all panels corresponds to noninfected cell autofluorescence.

FIG. 5

Identification of virus proteins associated with the cell membrane. Plasma cell membranes of MA104 cells infected with the RF strain of rotavirus (MOI = 10) were biotinylated at 6 h p.i. Then, cells were lysed as described in Materials and Methods, and biotinylated proteins were precipitated by streptavidin-agarose beads. Complexes from biotinylated infected cells (Inf∗.) or mock infected biotinylated cells (M.Inf∗.) or nonbiotinylated infected cells (inf.) were eluted by boiling in denaturing buffer sample and separated by SDS-PAGE (10% acrylamide; MOPS-Tris Novex system). Controls run on the same gel consisted of purified TLPs, and a total-cell lysate from infected cells (Total). Identical gels were blotted on a PVDF membrane and immunostained with polyclonal antibody 8148F directed against structural viral proteins (A), with MAb 5.73 directed against spike viral protein VP4 (B), or with MAb 164E22 directed against VP2 (C). Blots were revealed with anti-rabbit or anti-mouse alkaline phosphatase conjugate, respectively.

FIG. 6

Accessibility of viral proteins at the cell membrane. Cell monolayers were infected with rotavirus strain RF at an MOI of 10 (Inf.) or mock infected (M.Inf.). At 6 h p.i., an aliquot of infected cells was treated for 15 min with 200 μg of trypsin per ml (Inf.+Trypsin) at room temperature, and another aliquot was not treated (Inf.). Then, surface proteins were biotinylated, and aliquots of cell lysate corresponding to 10⁶ cells were incubated overnight with specific antibodies. Complexes were immunoprecipitated by protein A-Sepharose and boiled in denaturing sample buffer for 5 min. Proteins were separated by SDS-PAGE (10% acrylamide; Bis-Tris Novex system) and blotted on a PVDF membrane, and virus proteins coupled to biotin were revealed by a streptavidin-alkaline phosphatase reaction. Immunoprecipitation by anti-VP2 164E22 (lane 1), anti-VP4 5.73 (lane 2), anti-RF 8148 (lane 3), and anti-VP7 M60 (lane 4). Lanes M and TLP correspond to molecular weight markers and biotinylated TLP, respectively.

FIG. 7

Laser confocal microscopy of MA104 cells infected with bovine rotavirus or COS-7 cells expressing VP4-GFP chimera. MA104 cells were grown on slides, infected at an MOI of 1, fixed 6 h later with 2% PFA, and finally permeabilized with 1% Triton X-100. Cytoplasmic viral antigens were immunostained with MAb RV138, specific to VP6 (A) and MAb 7.7, specific to VP4 (B, D, and F), followed by an anti-mouse IgG conjugated to Alexa-488 (green). Microtubules were stained with an anti-β-tubulin MAb conjugated to CY 3 (red) (E to F). In panel F, both stains are superimposed, and colocalization appears in yellow. COS-7 cells fixed 48 h posttransfection with a plasmid directing the expression of the chimeric VP4-GFP protein were directly observed by confocal microscopy with the same excitation and emission wavelength used above for Alexia-488 (C). Bar = 20 μm.

FIG. 8

Effect of nocodazole on the localization of VP4. Cells were grown on slides, infected at an MOI of 1, not treated (A) or treated with 10 μg of nocodazole per ml (B), fixed 6 h later with 2% PFA, and finally permeabilized with 1% Triton X-100. Cells immunostained with anti-VP4 MAb 7.7 as described above were observed by confocal microscopy. Bar = 20 μm.