Characterization of a monoclonal antibody directed to the surface of MA104cells that blocks the infectivity of rotaviruses

Rhesus rotavirus (RRV) binds to sialic acid residues on the surface of targ et cells, and treatment of these cells with neuraminidase greatly reduces v irus binding with the consequent reduction of infectivity. Variants that ca n efficiently infect neuraminidase-treated cells have been isolated, indica ting that attachment to sialic acid is not an essential step for animal rot aviruses to infect cells. To identify and characterize the neuraminidase-re sistant receptor for rotaviruses, we have isolated a hybridoma that secrets a monoclonal antibody (MAb) (2D9) that specifically blocks the infectivity of wild-type (wt) RRV and of its sialic acid-independent variant nar3, in untreated as well as in neuraminidase-treated cells. The infectivity of a h uman rotavirus was also inhibited, although to a lesser extent. MAb 2D9 blo cks the binding of the variant to MA104 cells, while not affecting the bind ing of wt RRV; in addition, this MAb blocked the attachment of a recombinan t glutathione S-transferase (GST)-VP5 fusion protein, but did not affect th e binding of GST-VP8. Altogether these results suggest that MAb 2D9 is dire cted to the neuraminidase-resistane receptor. This receptor seems to mediat e the direct attachment of the variant to the cell, through VP5, while the receptor is used by wt RRV for a secondary interaction, after its initial b inding to sialic acid, through VP8. MAb 209 interacts specifically with the cell surface by indirect immunofluorescence, immunoelectron microscopy, an d FAGS. By a solid-phase immunoisolation technique, MAb 2D9 was found to re act with three proteins of ca. 47 55, and 220 kDa, which might form a compl ex. (C) 2000 Academic Press.