Abstract
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.
Highlights
The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A
The majority of the viruses studied to date use host-encoded glycosyltransferases (GTs)3 and glycosidases located in the endoplasmic reticulum and Golgi apparatus to add and remove N-linked sugar residues from virus glycoproteins either co-translationally or shortly after translation of the protein [2,3,4,5,6]
Gene a071r is predicted to encode a GT that attaches a semiconserved ␣-D-Rha to O-3 of the L-Fuc located in the N-glycan core region (Fig. 4)
Summary
PBCV-1 encodes six GTs, a small number when compared with the complexity of its N-glycans One solution to this paradox is that one or more of the six GTs might have multiple functional domains, making this restricted repertoire of enzymes sufficient to synthesize these structures. Several spontaneous mutants (or antigenic variants) in a064r have been isolated, and their MCPs migrate faster on SDS-PAGE compared with the MCP of PBCV-1, suggesting that the glycan portion of the capsid protein is altered. These PBCV-1 spontaneous mutants are divided into six antigenic classes, each denoted with a letter, based on their differential reaction to five different polyclonal antibodies [12, 17]. Using genetic and glycan structure evidence, we identify four of the putative GT activities involved in the synthesis of the glycan(s)
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