N-Linked Glycan Sites on the Influenza A Virus Neuraminidase Head Domain Are Required for Efficient Viral Incorporation and Replication

Abstract

N-linked glycans commonly contribute to secretory protein folding, sorting and signaling. For enveloped viruses such as the influenza A virus (IAV), the addition of large N-linked glycans can also prevent access to epitopes on the surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N). Sequence analysis showed that in the NA head domain of H1N1 IAVs three N-linked glycosylation sites are conserved and that a fourth site is conserved in H3N2 IAVs. Variable sites are almost exclusive to H1N1 IAVs of human origin, where the number of head glycosylation sites first increased over time and then decreased with and after the introduction of the 2009 pandemic H1N1 IAV of Eurasian swine origin. In contrast, variable sites exist in H3N2 IAVs of human and swine origin, where the number of head glycosylation sites has mainly increased over time. Analysis of IAVs carrying N1 and N2 mutants demonstrated that the N-linked glycosylation sites on the NA head domain are required for efficient virion incorporation and replication in cells and eggs. It also revealed that N1 stability is more affected by the head domain glycans, suggesting N2 is more amenable to glycan additions. Together, these results indicate that in addition to antigenicity, N-linked glycosylation sites can alter NA enzymatic stability and the NA amount in virions. IMPORTANCE N-linked glycans are transferred to secretory proteins upon entry into the endoplasmic reticulum lumen. In addition to promoting secretory protein maturation, enveloped viruses also utilize these large oligosaccharide structures to prevent access to surface antigen epitopes. Sequence analyses of the influenza A virus (IAV) surface antigen neuraminidase (NA or N) showed that the conservation of N-linked glycosylation sites on the NA enzymatic head domain differs by IAV subtype (H1N1 vs H3N2) and species of origin, with human derived IAVs possessing the most variability. Experimental analyses verified that the N-linked glycosylation sites on the NA head domain contribute to virion incorporation and replication. It also revealed that the head domain glycans affect N1 stability more than N2, suggesting N2 is more accommodating to glycan additions. These results demonstrate that in addition to antigenicity, changes in N-linked glycosylation sites can alter other properties of viral surface antigens and virions.

Balancing the influenza neuraminidase and hemagglutinin responses by exchanging the vaccine virus backbone

“…After BPL inactivation and purification, the viruses were adjusted to equal total protein concentrations and analysed. On both Coomassie stained SDS-PAGE gels (Fig 2C ) and immunoblots (Fig 2D), bands at the expected molecular weight of oxidized NA (dimers) and reduced NA (monomers) [19,36] were more apparent for WSN N1-BR18 than PR8 N1-BR18 , indicating WSN N1-BR18 virions incorporate more NA. The increase in NA levels correlated with a decrease in HA content but did not affect the HA processing to HA1 and HA2 or the content of the viral nucleoprotein (NP) or matrix (M1) protein (Fig 2C and 2E).…”

“…During previous studies, we observed that single-gene reassortant IAVs using NAs from subtype 1 (N1) are more easily rescued with a backbone from the A/WSN/1933 (WSN) strain compared to others and produce high NA levels [19,28,29]. To investigate this observation in a more controlled manner, single-gene reassortant IAVs carrying N1 antigens from the WHO recommended CVVs A/California/07/2009 (N1-CA09) or A/Brisbane/02/2018 (N1-BR18) were rescued using the seven gene segment backbone from WSN and PR8 (Fig 1A).…”

“…The IAV genome is comprised of eight single-stranded influenza RNA gene segments, which encode for one or more viral proteins [18]. Several studies have used reverse genetics to change the virion incorporation of HA or NA by altering the promoter region, codons, or Nlinked glycosylation sites in their respective gene segment [7,[19][20][21]. Similar investigations have found that exchanging the internal gene segments can also affect the NA and HA amounts in virions [22][23][24][25], a trait supported by more mechanistic analysis of NA and HA expression in cells [26,27].…”

Balancing the influenza neuraminidase and hemagglutinin responses by exchanging the vaccine virus backbone

“…During previous studies, we observed that single-gene reassortant IAVs using NAs from subtype 1 (N1) are more easily rescued with a backbone from the A/WSN/33 (WSN) strain compared to others and produce high NA levels [19, 28, 29]. To investigate this observation in a more controlled manner, single-gene reassortant IAVs carrying N1 antigens form the WHO recommended CVVs A/California/07/2009 (N1-CA09) or A/Brisbane/02/2018 (N1-BR18) were rescued using the seven gene segment backbone from WSN and PR8 (Fig.…”

“…2C) and immunoblots (Fig. 2D), bands at the expected molecular weight of oxidized NA (dimers) and reduced NA (monomers) [19, 36] were more apparent for WSN N1-BR18 than PR8 N1-BR18 , indicating WSN N1-BR18 virions incorporate more NA. The increase in NA levels correlated with a decrease in HA content but did not affect the efficiency of HA processing to HA1 and HA2 or the content of the viral nucleoprotein (NP) or matrix (M1) protein (Figs.…”