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

Abstract

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.

Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans

“…Moreover, A111/114R would be the second three-domain protein encoded by PBCV-1 that is involved in glycan synthesis. Indeed, recent studies showed that PBCV-1 protein A064R (638 aa) has three functional domains; the first two are GTs (β-L-rhamnosyltransferase and α-L-rhamnosyltransferase, respectively) and the third is a methyltransferase that methylates O-2 of the terminal α-L-Rha residue [ 30 ].…”

NDP-rhamnose biosynthesis and rhamnosyltransferases: building diverse glycoconjugates in nature

“…Although the glycobiology of viruses is less understood than eukaryotes or bacteria, with many viruses hijacking host glycosylation machinery for capsid glycosylation, it has recently been shown that viruses do produce rhamnose glycoconjugates. The giant Chlorella virus, Paramecium bursaria chlorovirus PBCV-1, synthesises highly complex N-glycans decorated on the major capsid protein that contain both L-and D-Rha (57,58) and are the first reported viral species that produce both enantiomers (Figure 1). Mimivirus has also been found to contain high amounts of L-Rha in its fibers (59).…”

“…Very recently, Speciale et al biochemically characterized the rhamnosyltransferase activity of a multifunctional enzyme from the algal-infecting giant virus, PBCV-1 (58). In this work, the authors showed that A064R contains 3 domains; 2 of which have ß-1,4-and α-1,2rhamnosyltransferase activity, with the third responsible for 2-O-methyltransferase activity which installs the 2-OMe group on the terminal rhamnose residue (58). In this work, the authors used several synthetic lipo-oligosaccharides and constructs of A064R varying in length, together with HPLC analysis, to characterize the activity of each domain.…”

Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

“…PBCV-1-encoded gene a064r, that encodes a highly characterized glycosyltransferase with three domains involved in protein glycosylation: domain 1 has a β-L-rhamnosyltransferase activity, domain 2 has an α-L-rhamnosyltransferase activity, and domain 3 is a methyltransferase (MT) that decorates one position in the terminal α-L-rhamnose unit [32]. The mutant selection scheme was based on the observation that CA-4B mutants can be selected by rabbit polyclonal antiserum derived from serologically distinct PBCV-1 mutants that have a mutation in gene a064r [33], that in turn produces truncated surface glycans.…”