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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.

DNA amplification-restricted transcription-translation (DARTT), is based on DNA amplification by the polymerase chain reaction (PCR) and uses PCR to truncate protein-encoding DNA while adding transcriptional and translational initiation signals to the segment. The amplified DNA segments are transcribed into RNA and translated into protein in vitro and the synthesized proteins are used to define functional sites. DARTT was applied to rhesus rotavirus gene segment 4 cDNA in order to create a series of carboxyl-terminal truncations and new amino termini in the encoded VP4 capsid protein. The truncated VP4 polypeptides were tested for reaction with 11 VP4-specific neutralizing monoclonal antibodies to identify the minimum polypeptides required for antibody recognition. Monoclonal antibodies 2G4, M2, and M7, which neutralize a number of serologically distinct rotaviruses, required amino acids 247-474 of VP4 for binding. DARTT is potentially applicable to the identification of discontinuous epitopes and functional domains on a variety of proteins.