viruses Article Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans Eric Noel 1,2,† , Anna Notaro 3,4,† , Immacolata Speciale 3,5,† , Garry A. Duncan 1, Cristina De Castro 5,* and James L. Van Etten 1,6,* 1 Nebraska Center for Virology, University of Nebraska, Lincoln, NE 68583-0900, USA; [email protected] (E.N.); [email protected] (G.A.D.) 2 School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0118, USA 3 Department of Chemical Sciences, University of Napoli Federico II, Via Cintia 4, 80126 Napoli, Italy; [email protected] (A.N.); [email protected] (I.S.) 4 Information Génomique et Structurale, Centre National de la Recherche Scientifique, Aix-Marseille Université, UMR 7256, IMM FR3479, Institut de Microbiologie (FR3479), CEDEX 9, 13288 Marseille, France 5 Department of Agricultural Sciences, University of Napoli Federico II, Via Università 100, 80055 Portici, Italy 6 Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722, USA * Correspondence: [email protected] (C.D.C.); [email protected] (J.L.V.E.) † These authors contributed equally to this work. Abstract: The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of ge- netic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in Citation: Noel, E.; Notaro, A.; all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino Speciale, I.; Duncan, G.A.; De Castro, C.; Van Etten, J.L. Chlorovirus PBCV-1 acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the Multidomain Protein A111/114R Has hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, Three Glycosyltransferase Functions domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein Involved in the Synthesis of Atypical likely responsible for the attachment of three of the five conserved residues of the core region of this N-Glycans. Viruses 2021, 13, 87. complex glycan, and, if biochemically corroborated, it would be the second three-domain protein https://doi.org/10.3390/v13010087 coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum–Golgi glycosyla- Academic Editors: Jacques Le Pendu tion pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of and Göran Larson cellular organelles using virus-encoded enzymes. Received: 9 December 2020 Accepted: 8 January 2021 Keywords: glycosyltransferases; multidomain protein; N-glycan; chloroviruses; PBCV-1 Published: 10 January 2021 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- 1. Introduction nal affiliations. Glycosylation is an important post-translational modification that confers a diver- sity of structures and functions in both cell and virus biology. One of the most common forms of protein modification is N-linked glycosylation, in which a high mannose core is linked to the amide nitrogen of asparagine in the context of the conserved motif Asn–X– Copyright: © 2021 by the authors. Li- Ser/Thr. This attachment occurs early in protein synthesis, followed by a complex process censee MDPI, Basel, Switzerland. of trimming and remodeling of the oligosaccharide during transit through the endoplas- This article is an open access article mic reticulum (ER) and Golgi [1] producing glycoproteins with varying oligosaccharide distributed under the terms and con- structures. Typically, viruses use host-encoded glycosyltransferases (GT) and glycosidases ditions of the Creative Commons At- N tribution (CC BY) license (https:// located in the ER and Golgi to add and remove -linked sugar residues to/from virus creativecommons.org/licenses/by/ glycoproteins either co-translationally or shortly after translation of the protein. The viral 4.0/). glycoproteins are then often transported to the host plasma-membrane where progeny Viruses 2021, 13, 87. https://doi.org/10.3390/v13010087 https://www.mdpi.com/journal/viruses Viruses 2021, 13, x FOR PEER REVIEW 2 of 15 Viruses 2021, 13, 87 cated in the ER and Golgi to add and remove N-linked sugar residues to/from virus2 ofgly- 14 coproteins either co-translationally or shortly after translation of the protein. The viral glycoproteins are then often transported to the host plasma-membrane where progeny viruses bud though the membrane, and so the viruses only become infectious as they leave viruses bud though the membrane, and so the viruses only become infectious as they leave the cell [[2].2]. However, thethe largelarge dsDNA dsDNA chloroviruses chloroviruses in in the the family familyPhycodnaviridae Phycodnaviridaeare are interesting interest- because,ing because, unlike unlike other other viruses, viruses, they they encode encode most, most, if not if all, not of all, the of machinery the machinery to glycosylate to glyco- theirsylate major their capsidmajor proteinscapsid protei (MCP)ns independent(MCP) independent of the cellular of the organellescellular organelles [3,4]. Furthermore, [3,4]. Fur- thethermore, process the occurs process in the occurs cytoplasm, in the andcytoplasm, infectious and viruses infectious are formed viruses inside are formed the cell inside prior tothe cell cell lysis. prior Theto cell prototype lysis. The chlorovirus, prototype chlorovirus, Paramecium Paramecium bursaria chlorella bursaria virus chlorella (PBCV-1), virus has(PBCV-1), a 330-kb has genome a 330-kb that genome is predicted that is topredic encodeted to as encode many as as 400 many proteins, as 400 manyproteins, of which many areof which unusual are for unusual a virus for [5], a including virus [5], at including least 17 related at least to 17 various related aspects to various of carbohydrate aspects of metabolismcarbohydrate [6 metabolism]. [6]. The PBCV-1 MCP (also referred to asas Vp54)Vp54) isis codedcoded byby genegene a430la430l,, andand thethe proteinprotein has four four glycosylation glycosylation sites sites [7]. [7 ].The The predominant predominant oligosaccharide oligosaccharide is a nonasaccharide is a nonasaccharide (Fig- (Figureure 1) with1) with several several unique unique features, features, includin includingg the the following: following: (i) its (i) itsstructure structure does does not not re- resemblesemble any any structure structure previously previously reported reported in the in thethree three domains domains of life of life[8]; [(ii)8]; the (ii) four the fourgly- glycoformscoforms are generated are generated by the by non-stochiom the non-stochiometricetric presence presence of two ofmonosaccharides, two monosaccharides, L-arab- L-arabinoseinose (Araf) and (Ara Df-mannose) and D-mannose (Man); (iii) (Man); the most (iii) theabunda mostnt abundant glycoform glycoform consists of consists nine neu- of ninetral monosaccharide neutral monosaccharide residues residuesorganized organized in a highly-branched in a highly-branched fashion; (iv) fashion; none (iv)of the none N- oflinked the Nglycans-linked is glycans attached is to attached a typical to Asn–X– a typical(Thr/Ser) Asn–X–(Thr/Ser) consensus site consensus in the protein site in the[9]; protein(v) the glycans [9]; (v) the are glycans attached are to attached the protein to the by protein a β-glucose by a β -glucose(Glc) linkage, (Glc) linkage,which is which rare in is rarenature in natureand has and only has been only reported been reported in glyc inoproteins glycoproteins from froma few a organisms few organisms [10–13]; [10– and13]; and(vi) the (vi) glycoform the glycoform contains contains a dimethylated a dimethylated rhamnose rhamnose (Rha) (Rha)as theas capping the capping residue residue of the ofmain the chain, main a chain, hyper-branched a hyper-branched fucose (Fuc) fucose residue (Fuc) residueand two andRha tworesidues Rha residueswith opposite with oppositeconfigurations. configurations. Figure 1. StructureStructure of ofthe the N-glycan N-glycan attached attached to the to chlorovirus the chlorovirus PBCV-1 PBCV-1 major major capsid capsid protein protein (Vp54).(Vp54). Monosaccharides Monosaccharides (Man (Man and and Ara Araf)f )connected connected by by dashed dashed lines lines are are non-stoichiometric non-stoichiometric sub- sub- stituents. The The larger larger black black box box encloses encloses the the conserved conserved pentasaccharide pentasaccharide core core structure structure common common to to all chloroviruses analyzed to date. The The inner inner red red box box indicates indicates the the tetrasaccharide tetrasaccharide structure structure of of the the N-glycan of the antigenic mutant chlorovirus PIL6PIL6 [[6].6]. Interestingly, allall thethe chloroviruseschloroviruses studiedstudied toto date,date, includingincluding thosethose withwith differentdifferent hosthost specificities,specificities, have the capsid protein N-glycosylated with other types of oligosaccharides; however, allall chloroviruseschloroviruses shareshare thethe