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 been shown to bind to MA-104 cells and human O type erythrocytes. To examine whether bacterially expressed VP8* binds to cellular components of MA-104 cells, the VP8* (aa 1-247) was expressed in E. coli and radiolabelled with 35S-methionine. The radiolabelled rVP8* was immunoprecipitated with antiserum to bovine rotavirus C486 (BRV). The rVP8* was found to bind to MA-104 cells and its binding was competed by BRV. To study the interaction between VP8* and receptors of erythrocytes, hemagglutination (HA) and hemagglutination inhibition (HI) assays were carried out using solubilized rVP8*. rVP8* showed HA which could be inhibited by antiserum to BRV. This interaction was also inhibited by gangliosides, demonstrating a sialic acid dependent interaction. To study the contribution of the C-terminal region of VP8* to HA, a number of approaches were used. First, a peptide spanning aa 230-247 was synthesized 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 dimerization of rVP8* and tVP8* was compared by Western-blotting following electrophoresis using native SDS-PAGE. The results indicated that antibody to aa 230-247 inhibits hemagglutination by preventing dimerization of VP8* which in turn allows the molecule to cause HA. To characterize 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 linkage-specific neuraminidase destroyed the ability of sialic acid of erythrocytes to interact with rVP8*, indicating that bovine rotavirus C486 binding requires an alpha 2-8 linkage but acetylation of the sialic acid is not necessary.
Lee
J
J
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon.
Yoo
D
D
Redmond
M J
MJ
Attah-Poku
S K
SK
van den Hurk
J V
JV
Babiuk
L A
LA
D000017:ABO Blood-Group System; D000818:Animals; D000906:Antibodies; D001667:Binding, Competitive; D002213:Capsid / Q000096:biosynthesis / Q000097:blood / Q000378:metabolism*; D036022:Capsid Proteins; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004912:Erythrocytes / Q000502:physiology / Q000821:virology*; D004926:Escherichia coli; D006385:Hemagglutination Inhibition Tests; D006386:Hemagglutination Tests; D006801:Humans; D016133:Polymerase Chain Reaction; D011817:Rabbits; D012401:Rotavirus / Q000502:physiology*; D014760:Viral Fusion Proteins / Q000378:metabolism
D000017:ABO Blood-Group System; D000906:Antibodies; D036022:Capsid Proteins; D017931:DNA Primers; D014760:Viral Fusion Proteins