Deglycosylation and truncation in the neuraminidase stalk are functionally equivalent in enhancing the pathogenicity of a high pathogenicity avian influenza virus in chickens

Abstract

Influenza A viruses with fewer amino acids in the neuraminidase (NA) stalk domain are primarily isolated from chickens rather than wild ducks, indicating that a shortened NA stalk is considered an adaptation marker of avian influenza viruses (AIVs) to chickens. Experimental passages of an H7N7 nonpathogenic AIV (rgVac2-P0) in chickens resulted in a highly pathogenic variant (Vac2-P3L4) with a 34-amino-acid deletion in the NA stalk, encompassing five potential N-glycosylation sites. To investigate how amino acid truncation and deglycosylation in the NA stalk contribute to increased pathogenicity, a virus with glycosylation-deficient mutations at these sites (rgVac2-P3L4/P0NAΔGlyco) was constructed. Contrary to expectations, chickens inoculated with rgVac2-P3L4/P0NAΔGlyco exhibited variable clinical outcomes, attributed to the genetic instability of the virus. A single mutation stabilized the virus, and the mutant (rgVac2-P3L4/P0NAΔGlyco-Y65H) resulted in higher pathogenicity compared with a virus with restored glycosylation (rgVac2-P3L4/P0NA-Y65H). Glycan occupancy analysis revealed 3-4 glycans at the five potential sites. In functional analysis, glycosylation-deficient mutants, similar to the short-stalk NA virus, showed significantly reduced erythrocyte elution activity. Additionally, mutational analysis indicated variable contributions of N-glycans to elution activity across the sites. Moreover, the functionally most contributing sites of the five potential N-glycosylation motifs were consistently included in the amino acid deletions of the stalk-truncated NA in N7-subtyped field isolates, despite the varying truncation position or length. These findings suggest that the loss of glycosylation is functionally equivalent to a reduction in amino acids, and it plays a crucial role in enhancing pathogenicity in chickens and affecting NA function.IMPORTANCEAvian influenza poses significant economic challenges to the poultry industry and presents potential risks to human health. Understanding the molecular mechanisms that facilitate the emergence of chicken-adapted avian influenza viruses (AIVs) from non-pathogenic duck-origin influenza viruses is crucial for improving AIV monitoring systems in poultry and controlling this disease. Amino acid deletions in the neuraminidase (NA) stalk domain serve as one of the molecular markers for AIV adaptation to Galliformes. This study highlights the critical role of N-glycosylation in the NA stalk domain in the pathogenesis of high pathogenicity avian influenza viruses in chickens. The findings propose a novel theory that the loss of glycosylation at the NA stalk domain, rather than a reduction in stalk length, is responsible for both NA function and increased virus pathogenicity in chickens.

Keywords: N-glycosylation; high pathogenicity avian influenza virus; neuraminidase; pathogenicity; site-specific glycan occupancy.

Fig 1

Survival of chickens intranasally inoculated with NA stalk mutant viruses. (A) Scheme of NA proteins of Vac2-P0NA, constructed from a membrane anchor, transmembrane domain, stalk domain, and globular head domains is shown. P0NA is a full-stalk NA, whereas P3L4NA, which was derived from several passages of rgVac2-P0 in chickens, has a 34-amino-acid deletion in the NA-stalk domain and one amino acid substitution in the transmembrane domain. P0NAΔGlyco was created by introducing N/Q mutations at the five potential N-glycosylation sites in the stalk domain. A series of viruses carrying either NA gene and accompanied by the L4 backbone were generated through reverse genetics in this study. (B) Six 6-week-old chickens were intranasally inoculated with 100 µL of each virus at 10^4.0 TCID₅₀ (50% tissue culture infectious dose) and observed for 14 days.

Fig 2

Survival of chickens and virus recovery from chickens intranasally inoculated with L4 mutants harboring Y65H substitution in the NA-stalk domain (A) Amino acid sequences of Vac2NA mutants with the Y65H mutation are shown. Recombinant viruses L4/P0NA-65H and L4/P0NAΔG-65H were generated using reverse genetics. (B) Six 6-week-old chickens were intranasally inoculated with 100 µL of each Y65H mutant at 10^4.0 TCID₅₀ and observed for 14 days. Survival of chickens inoculated with L4/P0NA and L4 is indicated with break lines. (C) Virus titers in the organs of chickens intranasally inoculated with each virus were evaluated 3 days post-inoculation. The dotted line indicates the threshold for virus titration (<1.8 log₁₀ TCID₅₀/g for organ samples and <0.8 log₁₀ TCID₅₀/g for blood). *Statistical analysis was performed using the Mann–Whitney U test between L4/P0NA-65H (light green) and L4/P0NAΔG-65H (orange). Virus titers below the detection limit were considered 1.8 log₁₀ TCID₅₀/g for tissue samples and 0.8 log₁₀ TCID₅₀/g for blood for statistical analysis.

Fig 3

Biological characteristics of Vac2 mutants with or without glycosylation in the NA-stalk domain. (A) Erythrocyte elution activity of Vac2/P0NA, Vac2/L4NA, and Vac2/P0NAΔG was assessed. These mutants harbor full-stalk NA, short-stalk NA, and glycan-deficient-stalk NA, respectively. (B) Sialidase activity of Vac2/P0NA, Vac2/L4NA, and Vac2/P0NAΔG to MUNANA was assessed. The initial reaction rate at each condition was plotted to calculate the Michaelis constant, K_m. The data were represented as the mean ± SEM of three independent experiments. (C) Erythrocyte elution activity of Vac2 mutants with partially glycosylated NA in the stalk was assessed. (D) Molecular weight of the NA proteins was compared to assess glycosylation of the NA-stalk domains under nontreated and PNGase F-treated conditions. Arrowhead indicates the aggregates of NA proteins of Vac2/P0NAΔG on the top of the resolving gel.

Fig 4

MS2 spectra of the peptide of P0NA with three glycans and mass chromatograms of precursor ions to identify site occupancy (A) MS2 spectra of the peptide of P0NA containing three glycans. (B) Molecular mass of b and y ion fragments derived from the peptide of P0NA. Red letters indicate b and y ion fragments detected in this analysis, whereas black letters refer to the calculated molecular weights based on the amino acid sequence. (C) The monoisotopic peak of MS1 corresponding to the less-glycosylated peptide was not observed. The blue arrow indicates the estimated molecular weight of the less-glycosylated peptide. (D) Mass chromatogram of the precursor ion corresponding to the less-glycosylated peptide was not detected.

Fig 5

MS2 spectrum of the peptide of P0NA with four glycans and mass chromatograms of precursor ions to identify site occupancy. (A) MS2 spectrum of the peptide of P0NA containing four glycans. (B) Molecular mass of b and y ion fragments derived from the peptide of P0NA with four glycans. Red letters indicate b and y ion fragments detected in this analysis, whereas black letters refer to the calculated molecular weights based on the amino acid sequence. (C) The monoisotopic peak of MS1 corresponding to the less-glycosylated peptide, which had a smaller molecular mass by 2.99 Da (m/z 0.997 [z = +3]; two blue arrows), was observed. (D) Mass chromatogram of precursor ions shows that the less-glycosylated peptide with three glycans (40.67 min; green) had a shorter LC retention time compared with the peptide with four glycans (40.98 min; red).

Fig 6

Glycosylation patterns of potential N-glycosylation sites in the NA-stalk domain of Vac2 mutants. (A) Amino acid sequences of the NA-stalk domain for the viruses constructed in this study are listed. Glycosylation patterns were summarized based on LC-MS/MS analysis. (B) Erythrocyte elution activity of Vac2 mutants with the Y65H substitution in the NA was assessed.

Fig 7

Overview of multiple potential N-glycosylation sites in the NA-stalk domain of N7NA proteins and patterns of amino acid truncation in natural isolates amino acid sequences of N7NA proteins covering the stalk domain were obtained from the Global Initiative on Sharing All Influenza Data (GISAID, https://gisaid.org/) and aligned with Vac2-P0NA and Vac2-P3L4NA. The sequences of stalk-truncated NAs are indicated, and the amino acid substitutions at the N-glycosylation sites in full-stalk NAs are summarized.