CHARACTERIZATION OF THE INTERACTION BETWEEN VP8 OF BOVINE ROTAVIRUS C486 AND CELLULAR-COMPONENTS ON MA-104 CELLS AND ERYTHROCYTES

Rotavirus VP8, the N-terminal trypsin cleavage product of VP4, has be en shown to bind to MA-104 cells and human O type erythrocytes. To exa mine whether bacterially expressed VP8 binds to cellular components o f MA-104 cells, the VP8 (aa 1-247) was expressed in E. coli and radio labelled with S-35-methionine. The radiolabelled rVP8 was immunopreci pitated with antiserum to bovine rotavirus C486 (BRV). The rVP8 was f ound to bind to MA-104 cells and its binding was competed by BRV. To s tudy the interaction between VP8 and receptors of erythrocytes, hemag glutination (HA) and hemagglutination inhibition (HI) assays were carr ied out using solubilized rVP8. rVP8* showed HA which could be inhibi ted by antiserum to BRV. This interaction was also inhibited by gangli osides, demonstrating a sialic acid dependent interaction. To study th e contribution of the C-terminal region of VP8 to HA, a number of app roaches were used. First, a peptide spanning aa 230-247 was synthesize d and antisera was raised against the peptide to see whether it could inhibit HA of rVP8. Second, a truncated form of VP8* (tVP8*: aa 1-229 ) was expressed to examine its hemagglutinating activity. Third, the d imerization of rVP8 and tVP8* was compared by Western-blotting follow ing electrophoresis using native SDS-PAGE. The results indicated that antibody to aa 230-247 inhibits hemagglutination by preventing dimeriz ation of VP8 which in turn allows the molecule to cause HA. To charac terize the interaction between the HA domain and sialic acid receptors , erythrocytes were treated with sialidases of different specificities . Arthrobacter ureafaciens, Clostridium perfringens and alpha 2-8 link age-specific neuraminidase destroyed the ability of sialic acid of ery throcytes to interact with rVP8, indicating that bovine rotavirus C48 6 binding requires an alpha 2-8 linkage but acetylation of the sialic acid is not necessary.