The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases

• Published in: The Journal of biological chemistry Vol. 294; no. 14; pp. 5688 - 5699
• Main Authors: Speciale, Immacolata, Duncan, Garry A., Unione, Luca, Agarkova, Irina V., Garozzo, Domenico, Jimenez-Barbero, Jesus, Lin, Sicheng, Lowary, Todd L., Molinaro, Antonio, Noel, Eric, Laugieri, Maria Elena, Tonetti, Michela G., Van Etten, James L., De Castro, Cristina
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.04.2019; American Society for Biochemistry and Molecular Biology
• Subjects: Antigens, Viral - genetics; Antigens, Viral - immunology; Antigens, Viral - metabolism; bioinformatics; capsid protein; Capsid Proteins - genetics; Capsid Proteins - immunology; Capsid Proteins - metabolism; Chlorella - genetics; Chlorella - metabolism; Chlorella - virology; DNA viruses; Glycobiology and Extracellular Matrices; glycosyltransferase; Glycosyltransferases - genetics; Glycosyltransferases - immunology; Glycosyltransferases - metabolism; molecular modeling; N-linked glycosylation; nuclear magnetic resonance (NMR); Paramecium bursaria chlorella virus 1 (PBCV-1); Phycodnaviridae - enzymology; Phycodnaviridae - genetics; Phycodnaviridae - immunology; Polysaccharides - genetics; Polysaccharides - immunology; Polysaccharides - metabolism; post-translational modification (PTM); post-translational modification (PTM); molecular modeling; glycosyltransferase; nuclear magnetic resonance (NMR); bioinformatics; Paramecium bursaria chlorella virus 1 (PBCV-1); capsid protein; N-linked glycosylation; DNA viruses
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA118.007182

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a β-l-rhamnosyltransferase activity, domain 2 has an α-l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal α-l-rhamnose (Rha) unit. The a075l gene encodes a β-xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved N-glycan core region. Last, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.