<i>N</i> -linked glycans on the stalk of influenza virus neuraminidase promote functional tetramer formation by compensating for local hydrophobicity

Enveloped virus surface antigens, such as influenza neuraminidase (NA), typically depend on <i>N-</i>linked glycans for assembly, trafficking in the host cell, and immune evasion. Here, we examined the function of the <i>N-</i>linked glycans on the NA stalk from H1N1 2009 pandemic (pdm09) viruses using reverse genetics coupled with a recombinant NA (rNA) analysis. Our results with the NA from A/Brisbane/02/2018 (H1N1) show that all five glycosylation sites in the stalk generally receive an <i>N-</i>linked glycan and that viral growth is largely unaffected by removing any of these sites individually. In contrast, viral growth decreased when we sequentially removed the stalk glycosylation sites, consistent with the observation that H1N1 pdm09 strains predominantly possess four or five stalk glycan sites in NA. Upon passage, the growth of the virus lacking the five stalk glycan sites significantly increased, and multiple substitutions were found that decreased the hydrophobicity near each mutated site. Reverse genetics experiments confirmed the polar stalk substitutions significantly improved viral growth and revealed an increase in the amount of NA dissociated from the virus, suggesting the stalk glycans or the hydrophobic regions protect against proteolysis. The polar substitutions also rescued the expression of a secreted recombinant NA (rNA) lacking the stalk glycan sites and significantly increased production of enzymatically active rNA that exceeded the wild-type version. These findings demonstrate that the stalk glycans promote functional NA tetramer formation by compensating for local hydrophobicity and that viral-based studies can be leveraged in the rational design of rNA antigens.IMPORTANCE<i>N-</i>linked glycans can play critical roles in viral glycoprotein maturation and immune evasion. The influenza virus glycoprotein neuraminidase (NA) possesses multiple <i>N-</i>linked glycan sites in the enzymatic head domain and stalk region that tether it to the viral surface. This study demonstrates that the stalk <i>N-</i>linked glycans contribute to viral fitness by compensating for local hydrophobicity to enable functional NA tetramer formation. Supporting this conclusion, our results show that sequential removal of the stalk glycan sites on a NA from a recent H1N1 strain leads to decreased viral growth that rapidly recovers following passage. The increased growth coincided with stalk mutations that reduced the hydrophobicity around the stalk glycan sites. We observed similar results with a secreted recombinant NA (rNA), and the amount of functional rNA produced with the polar substitutions exceeded the wild-type rNA, illustrating how viral-based studies can assist with the rational design of rNA antigens.