An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity

Abstract

Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1_low-1) and A/swine/Virginia/1814-2/2012 (pH1N1_low-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1_low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1_low-1 and pH1N1_low-2. pH1N1_low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1_low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

Importance: Neuraminidase (NA) is a sialidase that is one of the major surface glycoproteins of influenza A viruses and the target for the influenza drugs oseltamivir and zanamivir. NA is important as it releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. Mutations in the globular head domain that decrease enzymatic activity but confer resistance to NA inhibitors have been characterized; however, the importance of specific mutations in the stalk domain is unknown. We identified 66Y (N1 numbering), a highly conserved amino acid that was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

Keywords: influenza; neuraminidase; stalk.

FIG 1

Locations of mutations in the neuraminidase tetramer. (A) Location of mutations over the full length of the neuraminidase tetramer (side view). Mutations are shown in only one monomer. Horizontal black lines indicate the transmembrane region. (B) Top view of the neuraminidase tetramer, showing the locations of the mutations. (C and D) The amino acids in pH1N1-1 (C) and pH1N1_low-1 (D) at position 19, in the transmembrane domain, were modeled, showing their orientation within the tetramer. (E and F) The amino acids in pH1N1-1 (E) and pH1N1_low-1 (F) at position 66, in the stalk domain, were modeled, showing their orientation within the tetramer.

FIG 2

Mutagenesis of pH1N1_low neuraminidases revealed the importance of amino acid 66. (A) Mutagenesis of the pH1N1_low-1 neuraminidase (NA) showed that 66Y was required for the rescue of NA enzymatic activity. Relative to the enzymatic activity of pH1N1-1 NA, 66Y alone rescued 21.94 ± 0.838% of activity and all 5 mutations rescued 90.62 ± 6.46% of activity. 66Y with 19M, 232A, 248D, or 436I rescued 66.64 ± 4.81%, 37.04 ± 3.06%, 63.32 ± 3.23%, or 29.75 ± 3.02% of activity, respectively. (B) Mutagenesis of the pH1N1_low-2 NA revealed that 62V or 382G did not appreciably increase NA activity, while the quadruple mutant containing 19M, 232A, 248D, and 436I rescued 95.97 ± 7.39% of activity relative to that seen with pH1N1-1 NA. Data points represent the results of three independent experiments. Statistical significance was determined based on comparisons to the wild-type NA pH1N1_low-1 protein. **, P < 0.01.

FIG 3

66Y increased protein accumulation in transfected baby hamster kidney cells. (A) The intensity of the neuraminidase (NA) band of pH1N1-1 was greater than that of pH1N1_low-1 but similar to that of pH1N1_low-2. Introduction of 19M, 232A, 248D, or 436I into pH1N1_low-1 NA did not restore the band intensity to that of pH1N1-1, but the introduction of 66Y alone or all five mutations rescued band intensity. Introduction of 62V or 382G into pH1N1_low-2 NA did not increase band intensity. Introduction of 19M, 232A, 248D, and 436I into pH1N1_low-2 NA increased band intensity. (B) The band intensities were normalized to that of pH1N1-1. Data in panel B represent the means ± standard errors of the results of n = 3 transfections. NA was detected using an anti-HA tag antibody, and β-actin was used as loading control.

FIG 4

66Y increased the stability of pH1N1_low-1 neuraminidase, as measured by pulse-chase. The intensities of the neuraminidase (NA) bands at 0 and 3 h postchase (A) were compared and expressed in a bar graph (B). The intensities of the bands produced by pH1N1_low-1 NA containing 66Y at 3 h postchase were greater than those seen at 0 h postchase. The intensity of bands produced by pH1N1_low-1 NA containing 19M or 248D only at 3 h postchase were similar to those seen at 0 h postchase. Data in panel B represent the means ± standard errors of the results of n = 3 transfections.

FIG 5

66Y facilitated infection in the presence of human mucus. Reverse genetics (rg) analysis of 6+2 viruses on an A/WSN/33 (H1N1) background and containing the hemagglutinin (HA) and neuraminidase (NA) genes of pH1N1_low-1 and the D66Y mutation in NA showed increased infectivity of Madin-Darby canine kidney (MDCK) cells in the presence of human mucus compared to rg pH1N1_low-1 (A, C, G, and H). The infectivity of rg pH1N1_low-1 was also less than that of pH1N1-1 in the presence of mucus (panels A and B, respectively). In the absence of mucus, the infectivities of these viruses were similar to those of each other and to that of the rg pH1N1_low-1 virus (D, E, F, J, and K). The infectivity of rg pH1N1_low-1 was also less than the infectivity of the rg virus containing the HA of pH1N1_low-1 and the NA of pH1N1-1 in the presence of mucus (panels A and I, respectively) but similar in the absence of mucus (panels D and L, respectively). Scale bars, 100 μm. Blue, DAPI nuclear stain; green, influenza nucleoprotein. Images are representative of the results of three replicates of each experimental condition.

FIG 6

66Y alone or with 19M or 248D increased viral infectivity in the presence of mucus. Reverse genetics (rg) analysis of 6+2 viruses on an A/WSN/33 (H1N1) background and containing the HA and NA of pH1N1_low-1 and the D66Y mutation in NA showed increased infectivity of Madin-Darby canine kidney (MDCK) cells in the presence of human mucus compared to rg pH1N1_low-1. Viruses with D66Y and V19M or N248D infected the greatest percentage of cells. Viruses containing D66Y alone or the rg virus containing the HA of pH1N1_low-1 and the NA of pH1N1-1 infected similar percentages of cells but infected fewer cells than viruses containing 66Y and either 19M or 248D. **, P < 0.01; ***, P < 0.001 (compared to the mean infectivity of rg pH1N1_low-1 or pH1N1-1). Three monolayers were inoculated with each virus in the presence of human mucus. Each data point represents the mean percentage of cells infected in four representative fields.