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Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of
J J Meulenberg1, A P van Nieuwstadt, A van Essen-Zandbergen
1Institute for Animal Science and Health, Lelystad, The Netherlands. J.J.Meulenberg@id.dlo.nl
Abstract:
GP4 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 GP4 protein expressed by LV and the recombinant GP4 protein expressed in a Semliki Forest virus expression system. However, these MAbs did not react with the GP4 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 GP4 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 GP4 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 GP4. The neutralization domain of GP4, described here, is the first identified for LV.
Insights
Researchers identified the primary neutralization domain of the GP4 glycoprotein in Lelystad virus (LV). This domain, crucial for antibody binding, is located between amino acids 40-79 and shows significant variability across different LV isolates.
Area of Science:
- Virology
- Immunology
- Structural Biology
Background:
- Lelystad virus (LV) is an important pathogen, and its structural glycoproteins play a role in viral infectivity and host immune response.
- GP4 is a minor structural glycoprotein of LV, encoded by ORF4, with potential implications in viral neutralization.
Purpose of the Study:
- To characterize the GP4 glycoprotein of Lelystad virus (LV).
- To identify and map the epitope recognized by neutralizing monoclonal antibodies (MAbs).
- To investigate the variability of the GP4 neutralization domain across different LV isolates.
Main Methods:
- Immunoprecipitation and Western blotting to determine GP4 molecular mass and post-translational modifications.
- Expression of recombinant GP4 protein using a Semliki Forest virus system.
- Generation of chimeric constructs between LV and VR2332 ORF4 sequences.
- Epitope mapping using monoclonal antibody reactivity against chimeric proteins and synthetic peptides.
Main Results:
- GP4 exhibited different molecular masses (28 and 31 kDa) due to N-glycan modifications.
- A panel of 15 neutralizing MAbs reacted with LV GP4 but not with the U.S. isolate VR2332 GP4.
- Chimeric constructs localized the MAb binding site to amino acids 40-79 of LV GP4.
- The core neutralization epitope was mapped to amino acids 59-67 (SAAQEKISF).
- Sequence comparison revealed that the 40-79 region is the most variable part of GP4.
Conclusions:
- The study identified the first neutralization domain of the LV GP4 glycoprotein.
- This domain, spanning amino acids 40-79, is a key target for neutralizing antibodies.
- The high variability within this domain suggests potential immune evasion strategies by LV and has implications for vaccine development.