Expanding the Occurrence of Polysaccharides to the Viral World: The Case of Mimivirus.

The general perception of viruses is that they are small in terms of size and genome, and that they hijack the host machinery to glycosylate their capsid. Giant viruses subvert all these concepts: their particles are not small, and their genome is more complex than that of some bacteria. Regarding glycosylation, this concept has been already challenged by the finding that Chloroviruses have an autonomous glycosylation machinery that produces oligosaccharides similar in size to those of small viruses (6-12 units), albeit different in structure compared to the viral counterparts. We report herein that Mimivirus possesses a glycocalyx made of two different polysaccharides, now challenging the concept that all viruses coat their capsids with oligosaccharides of discrete size. This discovery contradicts the paradigm that such macromolecules are absent in viruses, blurring the boundaries between giant viruses and the cellular world and opening new avenues in the field of viral glycobiology.

Chlorovirus PBCV-1 protein A064R has three of the transferase activities necessary to synthesize its capsid protein N-linked glycans.

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 ß-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)-ß-l-Rha fragment. Remarkably, this fragment can be attached to any xylose unit.

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

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 ß-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 ß-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.

The rare sugar N-acetylated viosamine is a major component of Mimivirus fibers.

The giant virus Mimivirus encodes an autonomous glycosylation system that is thought to be responsible for the formation of complex and unusual glycans composing the fibers surrounding its icosahedral capsid, including the dideoxyhexose viosamine. Previous studies have identified a gene cluster in the virus genome, encoding enzymes involved in nucleotide-sugar production and glycan formation, but the functional characterization of these enzymes and the full identification of the glycans found in viral fibers remain incomplete. Because viosamine is typically found in acylated forms, we suspected that one of the genes might encode an acyltransferase, providing directions to our functional annotations. Bioinformatic analyses indicated that the L142 protein contains an N-terminal acyltransferase domain and a predicted C-terminal glycosyltransferase. Sequence analysis of the structural model of the L142 N-terminal domain indicated significant homology with some characterized sugar acetyltransferases that modify the C-4 amino group in the bacillosamine or perosamine biosynthetic pathways. Using mass spectrometry and NMR analyses, we confirmed that the L142 N-terminal domain is a sugar acetyltransferase, catalyzing the transfer of an acetyl moiety from acetyl-CoA to the C-4 amino group of UDP-d-viosamine. The presence of acetylated viosamine in vivo has also been confirmed on the glycosylated viral fibers, using GC-MS and NMR. This study represents the first report of a virally encoded sugar acetyltransferase.

The Autonomous Glycosylation of Large DNA Viruses.

Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production.

Giant virus Megavirus chilensis encodes the biosynthetic pathway for uncommon acetamido sugars.

Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways.

Characterization of a UDP-N-acetylglucosamine biosynthetic pathway encoded by the giant DNA virus Mimivirus.

Mimivirus is a giant DNA virus belonging to the Megaviridae family and infecting unicellular Eukaryotes of the genus Acanthamoeba. The viral particles are characterized by heavily glycosylated surface fibers. Several experiments suggest that Mimivirus and other related viruses encode an autonomous glycosylation system, forming viral glycoproteins independently of their host. In this study, we have characterized three Mimivirus proteins involved in the de novo uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) production: a glutamine-fructose-6-phosphate transaminase (CDS L619), a glucosamine-6-phosphate N-acetyltransferase (CDS L316) and a UDP-GlcNAc pyrophosphorylase (CDS R689). Sequence and enzymatic analyses have revealed some unique features of the viral pathway. While it follows the eukaryotic-like strategy, it also shares some properties of the prokaryotic pathway. Phylogenetic analyses revealed that the Megaviridae enzymes cluster in monophyletic groups, indicating that they share common ancestors, but did not support the hypothesis of recent acquisitions from one of the known hosts. Rather, viral clades branched at deep nodes in phylogenetic trees, forming independent clades outside sequenced cellular organisms. The intermediate properties between the eukaryotic and prokaryotic pathways, the phylogenetic analyses and the fact that these enzymes are shared between most of the known members of the Megaviridae family altogether suggest that the viral pathway has an ancient origin, resulting from lateral transfers of cellular genes early in the Megaviridae evolution, or from vertical inheritance from a more complex cellular ancestor (reductive evolution hypothesis). The identification of a virus-encoded UDP-GlcNAc pathway reinforces the concept that GlcNAc is a ubiquitous sugar representing a universal and fundamental process in all organisms.

Effects of microRNAs on fucosyltransferase 8 (FUT8) expression in hepatocarcinoma cells.

Fucosyltransferase 8 (FUT8) catalyzes the transfer of α1,6-linked fucose to the first N-acetylglucosamine in N-linked glycans (core fucosylation). Increased core fucosylation has been reported during hepatocarcinogenesis, in both cell-associated and secreted proteins. Accordingly, increased core fucosylation of α-fetoprotein and α1-antitrypsin is currently used as a diagnostic and prognostic indicator. The present study provides new evidences that FUT8 can be regulated also through miRNA-mediated mechanisms. Using microRNA/target prediction programs, we identified miR-122 and miR-34a seed regions in the 3' untranslated region (3'UTR) of FUT8. Then we used human and rodents hepatocarcinoma cell lines to evaluate the impact of transfection of miR-122 and miR-34a mimics on FUT8 mRNA and protein levels. This study demonstrated that forced expression of these miRNAs is able to induce a decrease of FUT8 levels and also to affect core fucosylation of secreted proteins. The ability of miR-122 and miR-34a to specifically interact with and regulate the 3'UTR of FUT8 was demonstrated via a luciferase reporter assay. Since miR-122 and miR-34a downregulation is a common feature in spontaneous human hepatocarcinoma, our finding that these miRNAs are able to target FUT8 3'UTR suggests that, together with transcriptional and other post-transcriptional systems, a miRNA-mediated mechanism could also be involved in the increased core fucosylation observed in liver tumors. Moreover, these findings also point out that miRNAs may be widely involved in the regulation of glycosylation machinery.

Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans.

The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a ß-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms.