Characterization of a monoclonal antibody directed to the surface of MA104 cells that blocks the infectivity of rotaviruses.
Rhesus rotavirus (RRV) binds to sialic acid residues on the surface of target cells, and treatment of these cells with neuraminidase greatly reduces virus binding with the consequent reduction of infectivity. Variants that can efficiently infect neuraminidase-treated cells have been isolated, indicating that attachment to sialic acid is not an essential step for animal rotaviruses to infect cells. To identify and characterize the neuraminidase-resistant 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 human rotavirus was also inhibited, although to a lesser extent. MAb 2D9 blocks the binding of the variant to MA104 cells, while not affecting the binding of wt RRV; in addition, this MAb blocked the attachment of a recombinant glutathione S-transferase (GST)-VP5 fusion protein, but did not affect the binding of GST-VP8. Altogether these results suggest that MAb 2D9 is directed to the neuraminidase-resistant receptor. This receptor seems to mediate 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 binding to sialic acid, through VP8. MAb 2D9 interacts specifically with the cell surface by indirect immunofluorescence, immunoelectron microscopy, and FACS. By a solid-phase immunoisolation technique, MAb 2D9 was found to react with three proteins of ca. 47, 55, and 220 kDa, which might form a complex.
López
S
S
Departamento de Génetica y Fisiología Molecular, Instituto de Biotecnología. susana@ibt.unam.mx
Espinosa
R
R
Isa
P
P
Merchant
M T
MT
Zárate
S
S
Méndez
E
E
Arias
C F
CF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology*; D000918:Antibody Specificity; D019921:Biotinylation; D002460:Cell Line; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008253:Macaca mulatta / Q000821:virology; D051379:Mice; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011991:Receptors, Virus / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012401:Rotavirus / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016601:RNA-Binding Proteins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C104282:nsp1 protein, Rotavirus; C079234:NS35 protein, rotavirus; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid