The general perception of viruses is that they are small in terms of size and genome,and that they hijackthe 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 sizet o those of small viruses (6-12 units), albeit different in structure compared to the viral counterparts.W er eport herein that Mimivirus possesses ag lycocalyx made of two different polysaccharides,n ow 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,b lurring the boundaries between giant viruses and the cellular world and opening new avenues in the field of viral glycobiology.

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 Megavirinae subfamily for which glycan-related genes were proposed to code for protein 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 Megavirinae 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 thirty-three genes to synthetize their fibril glycans (biosynthetic pathways for nucleotides sugars and glycosyltransferases). Second, they synthesize rare amino-sugars, usually restricted to bacteria and absent from their eukaryotic host. Finally, we showed that Megavirinae glycosylation is clade-specific and that Moumouvirus australiensis, a B-clade outsider, shares key features with Cotonvirus japonicus (clade E) and Tupanviruses (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 Megavirinae evolved such versatile glycosylation machinery.