RESUMO
Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.
Assuntos
Herpes Simples , Herpesvirus Humano 1 , Humanos , Herpesvirus Humano 1/genética , Herpes Simples/genética , Glicoproteínas/metabolismo , Queratinócitos/metabolismo , Polissacarídeos/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
The glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.