The rhesus rotavirus gene encoding protein VP3: location of amino acids involved in homologous and heterologous rotavirus neutralization and identification of a putative fusion region.

Abstract

The complete gene 4 nucleotide sequence was determined for rhesus rotavirus and each of 11 viral variants selected by neutralizing monoclonal antibodies. Gene 4 is 2362 bases in length and encodes a protein, VP3, of 776 amino acids with a calculated Mr of 86,500. A conserved trypsin cleavage site, located at amino acid 247, divides VP3 into VP8 and VP5. Neutralizing monoclonal antibodies directed at VP3 were used to select variants that escaped neutralization. Each variant contains a single gene 4 mutation that permits viral growth in the presence of the antibody. Variant mutations were identified in six distinct neutralization regions in VP8 and VP5. Five of the six neutralization regions were found in VP8. The VP8 regions were primarily associated with strain-specific or limited heterotypic rotavirus neutralization. One region was identified in VP5 by three monoclonal antibodies that neutralize a broad range of rotavirus serotypes. The VP5 neutralization region is largely hydrophobic and is similar to putative fusion sequences of Sindbis and Semliki Forest viruses.Rotavirus is a 70-nm icosahedral virus comprised of two capsid layers. The inner capsid, composed of proteins VP1, VP2, and VP6, contains an endogenous RNA-dependent RNA polymerase and 11 double-stranded RNA segments (1). Gene segments 4 and 9 encode the outer capsid proteins VP3 and VP7, respectively, of rhesus rotavirus (RRV) (2). VP7 (37 kDa) is a glycoprotein, and antibodies directed against VP7 neutralize the virus and specify the viral serotype (3-8). The other viral surface protein, VP3, is an 84-to 88-kDa nonglycosylated protein containing the viral hemagglutinin (9). In the presence of trypsin, VP3 is cleaved to VP5 (60 kDa) and VP8 (28 kDa), which results in enhancement of viral infectivity (10). Preliminary studies indicate that cleavage of VP3 mediates penetration of the virion into the cell (11). VP3 is also associated with restriction of virulence of certain rotavirus strains in mice (12) and humans (13). Antibodies directed at VP3 inhibit viral hemagglutination (14), neutralize the virus in vitro (14), and passively protect mice against heterologous rotavirus challenge in vivo (15). Further studies have demonstrated that VP3 effectively induces protective immunity in animals (16) and is immunogenic in young children (17).We have studied VP3 neutralization sites of the rhesus strain of simian rotavirus. RRV is serotypically identical to type 3 human rotavirus strains (18). Previously, we described a library of monoclonal antibodies (mAbs) directed against the RRV VP3 protein, which defines its functional and antigenic topography (22). mAbs directed at VP3 were used to delineate distinct neutralization domains and to demonstrate that VP3 contains both heterotypic and homotypic neutralizing epitopes. Neutralizing mAbs (N-mAbs) selecting these distinct domains were characterized by competitive binding studies and by their ability to recognize mutants that grew in the presence of N-mAbs.In this study, we have determined the full-len...

The localization and function of the neutralizing protein, VP4, in human rotavirus.

“…The mAb, which had both hemagglutination inhibiting and neutralizing activities, appeared to bind to the outer margin of double-shelled particles of human rotavirus, especially to the parts overlaying the tubelike channels, which seemed to be the "cover lid" of the tubelike channels that extruded radially from the core region. This binding point may be a putative fusion site of the virus (Mackow et al 1988) as well as a viral nucleus component ejection gate ). Rotavirus particles treated with the mAb were shown to be capable of binding to MA104 cells by ultra-thin section examination.…”

Identification of four VP4 serological types (P serotypes) of bovine rotavirus using viral reassortants

“…There was no reaction between sera that react with UK and B223 VP4s and viruses of any other P serotype. The neutralization observed between our two NCDV-specific VP4 antisera and non-bovine viruses is either known not to be substantiated by hybridization and sequence data in the case of RRV and 69M (Mackow et al, 1988;Qian & Green, 1991 ;Nishikawa et al, 1988), or, in the case of * Results are expressed as the magnitude of difference from antiserum titre with parental virus, i.e. 1, titre equal to or greater than with VP4 parental virus; 2, titre twofold less than with VP4 parental virus; 4, titre fourfold less than with VP4 parental virus; 8, titre eightfold less than with VP4 parental virus; 16, titre 16-fold less than with VP4 parental virus; -, titre > 16-fold less than with VP4 parental virus.…”

Analysis of the Newly Identified Neutralization Epitopes on VP7 of Human Rotavirus Serotype 1

“…Two major neutralization proteins on the outer capsid, VP4 and VP7, are encoded respectively by RNA segment 4 (Mason et al, 1983;Kalica et al, 1983) and segments 7, 8 or 9 depending on the strain (Greenberg et al, 1983a;Kalica et al, 1981;Ward et al, 1988). Most of the serotype-specific neutralization epitopes are located on VP7 (Greenberg et al, 1983bTaniguchi et al, 1985), while common antigens among different serotypes are mainly on VP4 (Mackow et aL, 1988b;Taniguchi et al, 1988b). Analysis of these neutralization antigens, especially the determination of amino acid positions that are critical for virus neutralization, provides valuable basic information for development of a rotavirus vaccine.…”