Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 life cycle

Enveloped viruses encounter multiple types of cellular glycans throughout their infectious cycles, eventually acquiring surface glycans themselves, largely determined by the cellular glycosyltransferase and modifying enzyme repertoire. The cellular glycans have the potential for modulating virus-host interactions, whereas the acquired glycans are expected to have broad functional and immunological implications with importance for the diagnosis and treatment of viral infections. Still, we only have limited tools to dissect the role of the individual classes of glycans in viral biology in a meaningful context. We have recently introduced genetic engineering as a powerful tool to dissect the importance of glycosylation in cell biology. Here we generated a glycoengineered cell library in human keratinocytes by CRISPR/Cas9 and ZFN gene editing composed of cells lacking different glycosyltransferases delineating human glycosylation pathways, and used it to address the importance of glycans in a clinically relevant model for herpes simplex virus type 1 (HSV-1). We propagated HSV-1 in cell lines with perturbations in the major classes of glycan structures, affecting the elongation and branching of N-linked and O-linked glycans, as well as elongation of glycosaminoglycans and glycosphingolipids. We also investigated the impact of site-specific O-glycosylation on the function of indispensable HSV-1 glycoproteins gB and gD. We find that distinct glycan structures are important at different stages of the virus life cycle; attachment, entry, and spread in the mutant cell lines as well as 3D organotypic skin equivalents. Our results show the importance of cellular glycosaminoglycans and glycolipids for attachment of HSV-1, N-glycans for entry and spread, and O-glycans for propagation. We further show that global changes in all elongated glycan structures of infection-competent virions had minimal effects on the early interactions with wild type cells. In contrast mutation of Ser or Thr residues at specific O-glycosylation sites directly affected glycoprotein surface expression and function. In conclusion, the data demonstrates the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures in herpesvirus biology. The method is applicable to other viral systems. ### Competing Interest Statement HHW owns stocks and is a consultant for and co-founder of EbuMab, ApS, Hemab, ApS, and GO-Therapeutics, Inc. All other authors declare no conflicts of interest.