Rotavirus spike protein VP4 is present at the plasma membrane and is associated with microtubules in infected cells

VP4 is an unglycosylated protein of the outer layer of the capsid of rotavi rus. It forms spikes that project from the outer layer of mature virions, w hich is mainly constituted by glycoprotein VP7. VP4 has been implicated in several important functions, such as cell attachment, penetration, hemagglu tination, neutralization, virulence, and host range. Previous studies indic ated that VP4 is located in the space between the periphery of the viroplas m and the outside of the endoplasmic reticulum in rotavirus-infected cells. Confocal microscopy of infected MA104 monolayers, immunostained with speci fic monoclonal antibodies, revealed that a significant fraction of VP4 was present at the plasma membrane early after infection. Another fraction of V P4 is cytoplasmic and colocalizes with beta-tubulin. Flow cytometry analysi s 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 a ccessible to antibodies. Biotin labeling of the infected cell surface monol ayer with a cell-impermeable reagent allowed the identification of the nonc leaved form of VP4 that was associated with the glycoprotein VP7. The local ization of VP4 was not modified in cells transfected with a plasmid allowin g the expression of a fusion protein consisting of VP4 and the green fluore scent protein. The present data suggest that VP4 reaches the plasma membran e through the microtubule network and that other viral proteins are dispens able for its targeting and transport.