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Significance Lassa virus is a highly pathogenic arenavirus that causes severe hemorrhagic fever in humans. Currently, there are no efficacious vaccines or treatments available to combat this pathogen. An important component of any vaccine candidate against Lassa virus will likely include the highly glycosylated glycoprotein complex presented on the virion surface. Here, we determine the composition of the Lassa virus glycome, revealing that the virus presents an abundance of glycans that are not biosynthetically processed to full maturity. Such underprocessed glycans form spatially distinct clusters, which shield the proteinous surface of the Lassa virus glycoprotein spike from the humoral immune response. These data are integral for the development of humoral-based vaccines that mimic the mature Lassa virion., Lassa virus is an Old World arenavirus endemic to West Africa that causes severe hemorrhagic fever. Vaccine development has focused on the envelope glycoprotein complex (GPC) that extends from the virion envelope. The often inadequate antibody immune response elicited by both vaccine and natural infection has been, in part, attributed to the abundance of N-linked glycosylation on the GPC. Here, using a virus-like−particle system that presents Lassa virus GPC in a native-like context, we determine the composite population of each of the N-linked glycosylation sites presented on the trimeric GPC spike. Our analysis reveals the presence of underprocessed oligomannose-type glycans, which form punctuated clusters that obscure the proteinous surface of both the GP1 attachment and GP2 fusion glycoprotein subunits of the Lassa virus GPC. These oligomannose clusters are seemingly derived as a result of sterically reduced accessibility to glycan processing enzymes, and limited amino acid diversification around these sites supports their role protecting against the humoral immune response. Combined, our data provide a structure-based blueprint for understanding how glycans render the glycoprotein spikes of Lassa virus and other Old World arenaviruses immunologically resistant targets.

Summary The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens., Graphical Abstract, Highlights • Quantitative, site-specific N-glycan analysis of a soluble HIV-1 Env trimer • A map of the extremes of simplicity and diversity at individual glycan sites • The fine structure of the mannose patch area of the Env trimer • How individual glycan sites influence HIV-1 Env-pseudovirus neutralization, Behrens et al. present detailed, quantitative, site-specific analyses of N-glycosylation sites of a soluble recombinant HIV-1 envelope glycoprotein trimer. The results highlight structural and antigenic details of the glycan shield that will be valuable for designing next-generation HIV-1 Env vaccines and understanding virus neutralization by broadly active antibodies.