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 ␤-L-rhamnosyltransferase activity, domain 2 has an ␣-L-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal ␣-L-rhamnose (Rha) unit. The a075l gene encodes a ␤-xylosyltrans-ferase 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, ␣-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.Structural proteins of many viruses, such as rhabdoviruses, herpesviruses, poxviruses, and paramyxoviruses, are glycosylated. Most virus glycoproteins are N-linked to Asn via N-acetylglucosamine (GlcNAc), whereas less frequent O-linked glycosylation also occurs (1). 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 (2-6). Post-translational glycosylation can aid in protein folding, protein stability, progression in the secretory pathway, and hostvirus interactions.One group of viruses that differs from the above scenario is the plaque-forming Chloroviruses (family Phycodnaviridae) that infect certain isolates of chlorella-like green algae (7). The viruses are divided into four groups, depending on the algal host infected: chloroviruses that infect Chlorella variabilis NC64A are referred to as NC64A viruses, those that infect C. variabilis

Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a β-l-rhamnosyltransferase; domain 2 is an α-l-rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α-l-rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α-l-Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N-glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α-l-Rha-(1→2)-β-l-Rha fragment. Remarkably, this fragment can be attached to any xylose unit.

Chlorella viruses produce N‐linked glycoproteins with carbohydrate moieties that differ in structure from all other N‐linked glycans. In addition, unlike most viruses, these organisms do not hijack the biosynthetic machinery of the host to make glycocoproteins; instead, they produce their own carbohydrate‐processing enzymes. A better understanding of the function and assembly of these fascinating and structurally‐unprecedented glycans requires access to probe molecules. This work describes the first synthesis of a chlorella virus N‐linked glycan, a highly branched hexasaccharide that contains the pentasaccharide present in all of the >15 structures reported to date. The target molecule includes a glucosyl‐asparagine linkage and a “hyperbranched” fucose residue in which all of the hydroxyl groups are glycosylated. Both convergent and linear approaches were investigated with the latter being successful in providing the target in 16 steps and 13 % overall yield.