Posttranslational processing and identification of a neutralization domain of the GP(4) protein encoded by ORF4 of Lelystad virus

GP(4) is a minor structural glycoprotein encoded by ORF4 of Lelystad virus (LV). When it was immunoprecipitated from cell lysates and extracellular virus of CL2621 cells infected with LV, it was shown to have an apparent molecular mass of approximately 28 and 31 kDa, respectively. This difference in size occurred because its core N-glycans were modified to complex type N-glycans during the transport of the protein through the endoplasmic reticulum and Golgi compartment. A panel of 15 neutralizing monoclonal antibodies (MAbs) reacted with the native GP(4) protein expressed by LV and the recombinant GP(4) protein expressed in a Semliki Forest virus expression system. However, these MAbs did not react with the GP(4) protein of U.S. isolate VR2332. To map the binding site of the MAbs, chimeric constructs composed of ORF4 of LV and VR2332 were generated. The reactivity of these constructs indicated that all the MAbs were directed against a region spanning amino acids 40 to 79 of the GP(4) protein of LV. Six MAbs reacted with solid-phase synthetic dodecapeptides. The core of this site consists of amino acids 59 to 67 (SAAQEKISF). Comparison of the amino acid sequences of GP(4) proteins from various European and North American isolates indicated that the neutralization domain spanning amino acids 40 to 79 is the most variable region of GP(4), The neutralization domain of GP(4), described here, is the first identified for LV.