Recombinant norovirus capsid protein VP1 (GII.4) expressed in H5 insect cells exhibits post-translational modifications with potential impact on lectin activity and vaccine design.

Abstract

Although surface proteins of most enveloped viruses are glycosylated, among non-enveloped viruses only few express glycoproteins in their capsid as infective virions. Noroviruses belong to the latter group and are known to express one major capsid protein (VP1) that lacks genuine glycosylation. In the context of vaccine development based on virus-like particles (VLPs) and in searches for food additives offering potential prophylactic or therapeutic applications an increasing number of reports refers to the use of VLPs that were produced as secretory products in insect cells. We asked the question whether recombinant VLPs (GII.4 Sydney, 2012) produced via the baculovirus vector in H5 insect cells may be glycosylated in the protruding domains that are involved in receptor binding and immune reactivity. Mass spectrometric analysis of tryptic VP1 peptides prior to and after beta-elimination Michael addition in 70% ethylamine revealed Thr238, and Ser519 in the P1 domain, and Thr350, Thr369, Thr371, and Thr381 in the P2 domain as modified. Thr65, Ser67, and Thr350 were revealed by liquid chromatography-mass spectrometry to carry HexNAc or Hex-HexNAc modifications, respectively. Monosaccharide analysis by gas chromatography-mass spectrometry confirmed the presence of GlcNAc on VLP protein, whereas immunoassays with lectins and antibodies demonstrated O-linked GlcNAc on VP1 protein. Post-translational modifications of virus capsid proteins may contribute to a modulation of immunodominant surface epitopes and need to be considered in anti-norovirus vaccine design. Some modifications are located near amino acid side chains involved in the binding of blood group active sugar receptors.