Giant viruses of the subfamily possess biosynthetic pathways to produce rare bacterial-like sugars in a clade-specific manner.

The recent discovery that giant viruses encode proteins related to sugar synthesis and processing paved the way for the study of their glycosylation machinery. We focused on the proposed subfamily, for which glycan-related genes were proposed to code for proteins involved in glycosylation of the layer of fibrils surrounding their icosahedral capsids. We compared sugar compositions and corresponding biosynthetic pathways among clade members using a combination of chemical and bioinformatics approaches. We first demonstrated that glycosylation differs in many aspects from what was previously reported for viruses, as they have complex glycosylation gene clusters made of six and up to 33 genes to synthetize their fibril glycans (biosynthetic pathways for nucleotide-sugars and glycosyltransferases). Second, they synthesize rare amino-sugars, usually restricted to bacteria and absent from their eukaryotic host. Finally, we showed that glycosylation is clade-specific and that , a B-clade outsider, shares key features with (clade E) and (clade D). The existence of a glycosylation toolbox in this family could represent an advantageous strategy to survive in an environment where members of the same family are competing for the same amoeba host. This study expands the field of viral glycobiology and raises questions on how evolved such versatile glycosylation machinery.