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. 2020 Aug 31;94(18):e01210-20.
doi: 10.1128/JVI.01210-20. Print 2020 Aug 31.

H9N2 Influenza Virus Infections in Human Cells Require a Balance between Neuraminidase Sialidase Activity and Hemagglutinin Receptor Affinity

Yasuha Arai  1 , Emad Mohamed Elgendy  1   2 , Tomo Daidoji  1 , Madiha Salah Ibrahim  2 , Takao Ono  3 , Nongluk Sriwilaijaroen  4   5 , Yasuo Suzuki  5   6 , Takaaki Nakaya  1 , Kazuhiko Matsumoto  3 , Yohei Watanabe  7
Affiliations

H9N2 Influenza Virus Infections in Human Cells Require a Balance between Neuraminidase Sialidase Activity and Hemagglutinin Receptor Affinity

Yasuha Arai et al. J Virol. .

Abstract

Some avian influenza (AI) viruses have a deletion of up to 20 to 30 amino acids in their neuraminidase (NA) stalk. This has been associated with changes in virus replication and host range. Currently prevalent H9N2 AI viruses have only a 2- or 3-amino-acid deletion, and such deletions were detected in G1 and Y280 lineage viruses, respectively. The effect of an NA deletion on the H9N2 phenotype has not been fully elucidated. In this study, we isolated G1 mutants that carried an 8-amino-acid deletion in their NA stalk. To systematically analyze the effect of NA stalk length and concomitant (de)glycosylation on G1 replication and host range, we generated G1 viruses that had various NA stalk lengths and that were either glycosylated or not glycosylated. The stalk length was correlated with NA sialidase activity, using low-molecular-weight substrates, and with virus elution efficacy from erythrocytes. G1 virus replication in avian cells and eggs was positively correlated with the NA stalk length but was negatively correlated in human cells and mice. NA stalk length modulated G1 virus entry into host cells, with shorter stalks enabling more efficient G1 entry into human cells. However, with a hemagglutinin (HA) with a higher α2,6-linked sialylglycan affinity, the effect of NA stalk length on G1 virus infection was reversed, with shorter NA stalks reducing virus entry into human cells. These results indicate that a balance between HA binding affinity and NA sialidase activity, modulated by NA stalk length, is required for optimal G1 virus entry into human airway cells.IMPORTANCE H9N2 avian influenza (AI) virus, one of the most prevalent AI viruses, has caused repeated poultry and human infections, posing a huge public health risk. The H9N2 virus has diversified into multiple lineages, with the G1 lineage being the most prevalent worldwide. In this study, we isolated G1 variants carrying an 8-amino-acid deletion in their NA stalk, which is, to our knowledge, the longest deletion found in H9N2 viruses in the field. The NA stalk length was found to modulate G1 virus entry into host cells, with the effects being species specific and dependent on the corresponding HA binding affinity. Our results suggest that, in nature, H9N2 G1 viruses balance their HA and NA functions by the NA stalk length, leading to the possible association of host range and virulence in poultry and mammals during the evolution of G1 lineage viruses.

Keywords: H9N2 influenza virus; functional balance; hemagglutinin; host range; infection; neuraminidase stalk.

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Figures

FIG 1
FIG 1
Phylogenetic trees of the HA and NA genes of H9N2 G1 viruses isolated in the Middle East. Phylogenetic trees were reconstructed from the nucleotide sequences of the HA (A) and NA (B) genes of the Middle East reference strains and of the prototype G1 virus using the neighbor-joining method with 1,000 bootstrap replicates. The HA and NA trees were rooted to A/quail/Hong Kong/G1/1997 (H9N2). The three G1 variants isolated in this study (i.e., CL79, CL80, and CL83) with NA stalk deletions are marked with black circles. The CL80 HA and NA genes are representative of those of G1-A/B strains with an 8-amino-acid NA stalk deletion and were used to generate recombinant viruses for this study. The CL42 internal genes are representative of the internal genes of G1-A/B viruses in Egypt and were used to generate recombinant viruses for this study. The CL42, CL79, CL80, and CL83 strains are underlined and in bold font in these trees. CK, DK, QL, and TK in the virus strain names denote chicken, duck, quail, and turkey hosts, respectively.
FIG 2
FIG 2
Generation of G1 viruses with various NA stalk lengths. (A) Schematic of the modified CL80 NA with various stalk lengths and glycosylation in this study. The 8-amino-acid deletion (58-IERNITEI-63) in the NA stalk compared to the consensus sequences of the G1 lineage strains in Egypt, including CL42 (consensus H9N2), was found in three G1-A/B strains. NA stalk length differences are shown relative to that of NA-wt. The blue area in the NA boxes indicates an amino acid insertion. A blue underline in the stalk sequences marks residues 61 to 63, which are a potential NA glycosylation site in the consensus H9N2 and NA+8aa/gly strains. A dotted line in an NA box and a black dashed line with an underline in a stalk sequence mark an amino acid deletion. (B) Western blot analysis of the NA mutants with a mobility shift indicating glycosylation in the NA+8aa/gly mutant. A representative Western blot is shown. (C) Quantification of NA expression in 293T cells. The cells were transfected with plasmids expressing the indicated NAs, and cell lysates were analyzed by Western blotting using anti-NA antibody. Each data bar shows the mean ± SD from four independent experiments. nt, nucleotide; NS, no statistically significant difference.
FIG 3
FIG 3
Enzymatic activity of the NA stalk length mutants. (A) The sialidase activity of each NA stalk length mutant was measured using the soluble NA-XTD substrate. Virus samples were standardized to equivalent FFU and HAU titers. NA activities were calculated relative to that of NA-wt. Each data bar shows the mean ± SD from three independent experiments. *, P < 0.01. The schematic of the NA stalk in each strain, as shown in Fig. 2A, is shown at the left. (B and C) Enzyme kinetics of the NA stalk length mutants using the soluble MUNANA substrate. (B) Initial reaction velocity with standardized virus FFU titers. (C) Initial reaction velocity with standardized virus HAU titers. The kinetic data for each reaction were fit to the Michaelis-Menten equation by nonlinear fitting to determine the Michaelis-Menten constant (Km) and the maximum velocity of substrate cleavage (Vmax). Each data point is the mean ± SD from three independent experiments with duplicate samples.
FIG 4
FIG 4
Elution of NA stalk length mutants bound to chicken and turkey erythrocytes (RBCs). (A to C) Twofold dilutions of 128 HAU of each NA mutant were incubated at 4°C for 1 h with an equal volume of chicken erythrocytes (A), turkey erythrocytes (B), and turkey erythrocytes that had been treated with α2,3 neuraminidase (C). The samples were then transferred to 33°C, and their HA titers were assayed as a function of time. The data are expressed relative to the percentage of the initial HA titer at time zero at 4°C. Each data point is the mean ± SD from three independent experiments. The Sia expressed on each type of erythrocyte is shown in parentheses at the left axis. (D) The time when half of each virus eluted from each type of erythrocyte was calculated by fitting the HA elution data with the Boltzmann equation. The data are expressed relative to the results for NA-wt. Each data point is the mean ± SD from three independent experiments. NS, no statistically significant difference. A schematic of the NA stalk in each strain, as shown in Fig. 2A, is shown at the left.
FIG 5
FIG 5
Replication of NA stalk length mutants in an avian cell line and eggs. (A and C) Growth kinetics of NA mutants in avian cells and eggs. (A) Chicken DF-1 cells were infected with the indicated viruses at an MOI of 0.005 and cultured at 37°C for 96 hpi. (C) Nine-day-old embryonated chicken eggs (5 eggs per group) were inoculated with 1 × 105 FFU of the indicated viruses and incubated at 37°C for 72 hpi. The culture supernatants and allantoic fluids were harvested at the indicated times and assayed for the numbers of FFU to determine the progeny virus titers. (B and D) Relative virus yields at 48 hpi. The virus titers produced by the NA mutants in DF-1 cells (B) and eggs (D) are shown relative to the titers produced by NA-wt. Each data point is the mean ± SD from three independent experiments. *, P < 0.01. A schematic of the NA stalk in each strain, as shown in Fig. 2A, is shown at the left.
FIG 6
FIG 6
Replication of NA stalk length mutants in a human cell line and primary airway cells. (A and C) Growth kinetics of NA mutants in human Calu-3 cells (A) and primary human bronchial epithelial cells (C). The cells were infected with the indicated viruses at an MOI of 0.03 and cultured at 33°C for 96 hpi. The culture supernatants were harvested at the indicated times and assayed for the numbers of FFU to determine the progeny virus titers. (B and D) Relative progeny virus titers at 48 hpi. The virus titers produced by the NA mutants in Calu-3 cells (B) and primary human airway cells (D) are expressed relative to the titers produced by NA-wt. Each data point is the mean ± SD from three independent experiments. *, P < 0.01. A schematic of the NA stalk in each strain, as shown in Fig. 2A, is shown at the left.
FIG 7
FIG 7
Virulence and in vivo replication of NA stalk length mutants in mice. (A and B) BALB/c mice (6 mice per group) were inoculated intranasally with 105 (top) or 106 (bottom) FFU of NA-wt and or an NA mutant virus with the indicated NA stalk length. (A) The body weights of the infected mice were monitored daily for 14 dpi. The mean ± SD of the percentage of the initial body weight for each group of mice is shown. (B) Survival of the infected mice. Survival was calculated for all mice, including mice that were humanely sacrificed if they lost more than 30% of their body weight within a few days. A schematic of the NA stalk of each strain (as shown in Fig. 2A), the MLD50 of that strain, and its value relative to that for NA-wt (in parentheses) are shown at the right. (C) Virus titers at 4 dpi in the lungs of mice (5 mice per group) infected with 105 FFU of the indicated viruses. Each symbol marks the titer in an individual mouse. *, P < 0.01. (D) (Top row) Representative low-magnification photomicrographs at 4 dpi of hematoxylin- and eosin (HE)-stained sections of lungs from mice infected with the indicated viruses. (Bottom) Magnified views of the photomicrographs in the top row. The different NA stalk lengths in the viruses are shown above panel D.
FIG 8
FIG 8
Receptor binding specificity of the CL80 HA, CL42 HA, Em/R66 HA, and mutant HAs. (A to C) Binding assays of the indicated viruses to three receptor analogues: α2,6 SLN, α2,3 SLN, and sulfated α2,3 SLN (Su-α2,3 SLN). CL42 HA is representative of a G1 lineage HA with high α2,6 Sia binding. Each data point is the mean ± SD from three independent experiments.
FIG 9
FIG 9
Infectivity of G1 viruses with reassorted HA/NA in cultured cells. Semiconfluent monolayers of cells grown in 96-well plates were infected with G1 viruses carrying an NA mutant with a modified stalk and an HA from CL80 (left), CL42 (middle), or Em/R66 (right). The MOIs of these infections were equalized by FFU assays on MDCK cells. (A) MDCK cells; (B) DF-1 cells; (C) Calu-3 cells. After 1 h of adsorption and washing, the cells were overlaid with 1% methylcellulose in medium without trypsin, to prevent the release of progeny virus, and analyzed by immunofluorescence assays. Virus infections were assayed by counting virus antigen-positive cells and cell nuclei in the same field with an IN Cell analyzer automated microscope. Data are expressed as the mean ± SD from 14 independent experiments, with the results of individual experiments shown as open symbols. *, P < 0.01. A schematic of the NA stalk in each strain, as shown in Fig. 2A, is shown at the left.
FIG 10
FIG 10
Progeny virus release of NA stalk length mutants. (A) Representative transmission electron micrographs of thin sections of MDCK cells infected by the indicated viruses (low magnification). (B) High magnification of the micrographs in panel A, showing progeny virions budding from the cell surface and virus aggregates.
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