Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses

Abstract

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>>>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>>>dimer. AH N1 from pseudoparticles existed in the forms monomer>>dimer, but after insertion of 09s60, it existed in the forms dimer>>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.

Figure 1. Diagrams of HA/NA expression plasmids, GagPol-encoding plasmid, and CMV–GFP reporter plasmid, and transmission electron microscope observation.

A. HA/NA expression plasmids, HA/NA encoding sequences were inserted between the CMV promoter and poly A Tbgh (bovine growth hormone) tail. B. Murine leukemia virus GagPol-encoding plasmid, GagPol-encoding sequence was inserted between the CMV promoter and the Rabbit beta-globin G ploy A tail. C. CMV–GFP reporter plasmid, the EGFP sequence was inserted into the U3 and U5 sequence of murine leukemia virus. D. Transmission electron microscopy image of pseudoparticles.

Figure 2. Alignment of influenza virus A hemagglutinin (HA; upper) and neuraminidase (NA; lower) sequences to those of the 2009 pandemic H1N1 strain (2009H1N1).

Dots indicate residues that are identical to the corresponding residues in 2009H1N1, and dashes indicate deleted residues. Signal peptides are shown in bold type. Gray boxes indicate potential glycosylation sites, as predicted from the amino acid sequence. For HA, red residues represent the precursor cleavage site that links the functional HA1 and HA2 domains, and blue residues represent the binding site for the sialic acid receptor. For NA, conserved active site residues are highlighted in green, and the 20-aa insertion/deletion segment of interest in the stalk region is highlighted in yellow.

Figure 3. Influence of NAs on viral release.

The AH N1, AH N1+09s60, 09N1, and 09N1-s60 were combined with AH H5 or 09H1 HA to generate eight pseudoparticles, and the release efficiencies of the resulting pseudoparticles were assessed by quantitative PCR (n = 4).

Figure 4. NA and hemagglutination activities of pseudoparticles.

(A) NA assay. Pseudoparticles were normalized for RNA copy number and analyzed for NA activity using a chemiluminescent substrate (n = 4). (B) Hemagglutination assay. Pseudoparticles were serially diluted 1:2 in a 96-well plate. Hemagglutination activity is expressed as the mean HA titer (log2 HA units/50 µl) of each pseudoparticle (n = 4).

Figure 5. Infectivity of normalized pseudoparticles.

Infectivity is presented as the mean ± SD percentage of infected cells (n = 4). Blue bars, infectivity of pseudoparticles comprising native combinations of HA and NA (09H1::09N1 and AH H5::AH N1); gray bars, infectivity of mismatched pseudoparticles; white bars, infectivity of pseudoparticles in the presence of oseltamivir.

Figure 6. Western blot analysis of expression of NA in pseudoparticle producer 293 cells and NA incorporation into pseudoparticles.

(A) Pseudoparticles and their producer 293T cell lysates were analyzed for 09N1 and 09N1-s60 expression. Wild-type H1N1 virus A/California/7/2009 (300 ng) was used as a positive control. Mock, normal 293T cell lysate; pps, pseudoparticles. (B) Pseudoparticles and their producer 293T cell lysates were analyzed for AH N1 and AH N1+09s60 expression. Mock, normal 293T cell lysate; pps, pseudoparticles.

Figure 7. Western blot analysis of expression of HA in pseudoparticle producer 293 cells and HA incorporation into pseudoparticles.

(A) Pseudoparticles and their producer 293T cell lysates were analyzed for AH H5 expression. Wild-type H5N1 virus A/Vietnam/1194/2005 (300 ng) was used as a positive control. (B) Pseudoparticles and their producer 293T cell lysates were analyzed for 09H1 expression. Wild-type H1N1 virus A/California/7/2009 (300 ng) was used as a positive control.