In contrast to the rather well documented roles of N-linked glycans, there are very few known examples, where specific O-glycans participate in virus–host interaction. Among these, two sialylated O-glycans on HSV-1 gB have been identified that determine binding to cellular receptor paired immunoglobulin-like type 2 receptor α residing on immune cells (Wang et al. 2009; Arii et al. 2010; Bagdonaite et al. 2015). In addition, HSV-1 O-glycans are involved in a few other aspects of viral binding to the host cell, as deletion of the densely O-glycosylated region of attachment factor gC affects both the binding affinity to the cell surface, and the release of progeny virus via modulation of interactions with cell surface glycosaminoglycans (Altgarde et al. 2015). Another example includes the carbohydrate-dependent binding of filoviruses to the macrophage galactose lectin that is known to recognize GalNAc-O-glycans (Takada et al. 2004). In hepatitis C virus, the mutation of several putative O-glycosites has also been shown to decrease HCV E2 affinity for CD81, suggesting that O-glycans might be of a more general importance in mediating interaction with host cells (Falkowska et al. 2007). O-glycoproteomic analysis of Hendra virus glycoprotein G has recently sprouted the first, to the best of our knowledge, systematic functional analysis of known O-glycosylation sites, revealing a multitude of functions including attachment and entry to host cells (Colgrave et al. 2012; Stone et al. 2016). Importantly, most of the functions could also be identified in analogous O-glycosites of a closely related Nipah virus (Stone et al. 2016). Interestingly, only in Nipah virus a single O-glycosite significantly affected Ephrin B2 receptor binding (Stone et al. 2016). This is an important example of conserved O-glycan function between closely related viruses, and presents intriguing possibilities in the light of recently identified consistent O-glycosylation patterns of several herpesviruses. Besides confirming the HSV-1 O-glycosites involved in binding PILRα in vitro, a few other O-glycosites residing in protein regions expected to influence HSV-1 attachment via cell entry receptors nectin-1 and HVEM (Carfi et al. 2001; Krummenacher et al. 2005; Heldwein et al. 2006; Di Giovine et al. 2011; Gallagher et al. 2014), or VZV attachment to receptor insulin degrading enzyme (Berarducci et al. 2010) were identified (Bagdonaite et al. 2015, 2016). This is another example how MS-based approaches combined with structural knowledge can help narrow down a list of O-glycosite candidates for focused studies. In conclusion, glycans on viral entry proteins are widely used for modulation of receptor binding and entry, with both N-linked and O-linked glycans having the capacity to affect the interaction (Figure 2, top left panel). In some cases, though, it seems to be an effect of conformational stability, rather than direct interaction (Stone et al. 2016).