AI-driven structural informatics reveals diverse glycosylation machinery across double-stranded DNA viruses

Abstract Glycosylation is widespread in biology and shapes molecular recognition, yet the diversity and biosynthetic organization of viral glycosyltransferases (GTs) remain poorly understood. Here we integrate structural-alphabet-guided searches and structure similarity networks with genomic-neighbourhood and biosynthetic gene cluster embedding analyses to examine viral GT-A proteins. This approach reveals extensive structural diversity overlooked by sequence-based annotation, including previously uncharacterized viral GT lineages. Most strikingly, we identified a jumbo-phage locus encoding five tandem glycosyltransferases alongside hydroxymethyl-pyrimidine biosynthetic and sulfation-associated enzymes, suggesting a previously unrecognized form of viral DNA modification involving a sulfated pentasaccharide. Beyond these examples, predicted saccharide-associated loci span divergent double-stranded DNA viruses, from giant viruses to tailed phages, and occupy multiple regions of the reference biosynthetic landscape. Together, our findings uncover an underappreciated repertoire of virus-encoded glycosylation machinery and suggest that viral glycan biosynthesis encompasses diverse molecular contexts and potential biological functions.