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

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 ␤-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

Chloroviruses

“…The conclusion that the chlorovirus PBCV-1 MCP (protein Vp54, gene a430l) is glycosylated by a different mechanism than that used by other characterized viruses began from antibody studies in 1993 [212]. Rabbit polyclonal antiserum [213] particles inhibited virus plaque formation by agglutinating the virions. Spontaneously-derived antiserum-resistant, plaque-forming variants of PBCV-1 occur at a frequency of 10 −5 to 10 −6 .…”

“…PBCV-1 encodes at least eight putative glycosyltransferases (GTs), some of which, if not all, participate in glycosylating the virus Vp54 protein [199,213,216,217]. Ongoing experiments have identified the role of three PBCV-1-encoded GTs with four GT activities that are involved in the synthesis of the glycan ( Figure 10A).…”

“…Ongoing experiments have identified the role of three PBCV-1-encoded GTs with four GT activities that are involved in the synthesis of the glycan ( Figure 10A). Two of the three GTs were previously annotated as GTs but the third protein was only identified in a recent study [213].…”

“…and independent of the ER and Golgi apparatus [212,213]. For example, unlike viruses that acquire their surface glycoprotein(s) by budding through the plasma membrane and only become infectious during their release from the host, intact infectious PBCV-1 particles accumulate inside their host 30-40 min prior to virus release [35].…”

“…Most significantly, the PBCV-1 gene a064r encodes a protein with three domains: domain 1 has a β-l-rhamnosyltransferase activity [213,[216][217][218], domain 2 has an α-l-rhamnosyltransferase activity [213,217], and domain 3 encodes a methyltransferase that methylates the 2 position in the terminal α-l-rhamnose unit ( Figure 10B) [217]. In retrospect the a064r gene is interesting because in the late 1990s we tried to make recombinant viruses between different PBCV-1 anti-genetic variants to create wild-type viruses [216].…”