Use of a lambda gt11 expression library to localize a neutralizing antibody-binding site in glycoprotein E2 of Sindbis virus.
The Sindbis virus envelope contains two species of integral membrane glycoproteins, E1 and E2. These proteins form heterodimers, and three dimeric units assemble to form spikes incorporated into the viral surface which play an important role in the specific attachment of Sindbis virus to host cells. To map the neutralization epitopes on the surface of the virus, we constructed a lambda gt11 expression library with cDNA inserts 100 to 300 nucleotides long obtained from randomly primed synthesis on Sindbis virus genomic RNA. This library was screened with five different neutralizing monoclonal antibodies (MAbs) specific for E2 (MAbs 50, 51, 49, 18, and 23) and with one neutralizing MAb specific for E1 (MAb 33). When 10(6) lambda gt11 plaques were screened with each antibody, four positive clones that reacted with E2-specific MAb 23 were found. These four clones contained overlapping inserts from glycoprotein E2; the domain from residues 173 to 220 of glycoprotein E2 was present in all inserts, and we concluded that this region contains the neutralization epitope recognized by the antibody. No clones that reacted with the other antibodies examined were found, and we concluded that these antibodies probably recognize conformational epitopes not present in the lambda gt11 library. We suggest that the E2 domain from residues 173 to 220 is a major antigenic determinant of Sindbis virus and that this domain is important for virus attachment to cells.
Wang
K S
KS
Division of Biology, California Institute of Technology, Pasadena 91125.
Strauss
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D002478:Cells, Cultured; D002642:Chick Embryo; D000939:Epitopes / Q000032:analysis*; D015723:Gene Library; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009500:Neutralization Tests; D012845:Sindbis Virus / Q000235:genetics / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014759:Viral Envelope Proteins; C064950:glycoprotein E2, Sindbis virus