Influenza A viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice

Abstract

Influenza A viruses possess both hemagglutinin (HA), which is responsible for binding to the terminal sialic acid of sialyloligosaccharides on the cell surface, and neuraminidase (NA), which contains sialidase activity that removes sialic acid from sialyloligosaccharides. Interplay between HA receptor-binding and NA receptor-destroying sialidase activity appears to be important for replication of the virus. Previous studies by others have shown that influenza A viruses lacking sialidase activity can undergo multiple cycles of replication if sialidase activity is provided exogenously. To investigate the sialidase requirement of influenza viruses further, we generated a series of sialidase-deficient mutants. Although their growth was less efficient than that of the parental NA-dependent virus, these viruses underwent multiple cycles of replication in cell culture, eggs, and mice. To understand the molecular basis of this viral growth adaptation in the absence of sialidase activity, we investigated changes in the HA receptor-binding affinity of the sialidase-deficient mutants. The results show that mutations around the HA receptor-binding pocket reduce the virus's affinity for cellular receptors, compensating for the loss of sialidase. Thus, sialidase activity is not absolutely required in the influenza A virus life cycle but appears to be necessary for efficient virus replication.

FIG. 1

Structure of the NA gene of the 23ΔNA NA deletion mutant. (A) Schematic diagram of the genomic structure of the 23ΔNA NA gene (cRNA orientation). The 23ΔNA NA gene contains a 728-nucleotide deletion (from bases 442 to 1170) that removes a large portion of the NA gene coding sequence. This mutant also contains a mutation at base 110 that forms an in-frame TAG stop codon. (B) Potential gene products encoded by this gene. The stop codon at bases 109 to 111 results in termination of the NA coding sequence at codon 31. The remaining portion of the open reading frame corresponds to the six-amino-acid (aa) tail and 24 amino acids of the transmembrane region. wt, wild type.

FIG. 2

Sialidase activity of the parental NWS-G70c virus, 23ΔNA, and the sialidase-independent CK2-29 mutant. For each sample, virus (2 × 10⁴ PFU) was incubated for 1 h at 37°C in the presence of a fluorogenic sialidase substrate (4-methylumbelliferyl-α-d-N-acetylneuraminic acid) in triplicate. The fluorescence of released 4-methylumbelliferone was determined with a fluorometer (Labsystems Fluoroskan II), with excitation at 355 nm and emission at 460 nm. Standard errors for the triplicate samples (at the 95% confidence interval) for NWS-G70c were too small to present in the graph. The detectable sialidase activity in the 23ΔNA NA deletion mutant results from sialidase activity of the N2 NA supplied in trans from 23-1i cells.

FIG. 3

Fetuin-binding affinity of NWS-G70c and its NA deletion variants. The viruses were adsorbed in the wells of plastic microplates and allowed to bind either peroxidase-labeled fetuin (Fet-HRP) (A) or rabbit anti-WSN antibodies, followed by peroxidase-conjugated anti-rabbit IgG antibodies (B). Levels of bound fetuin or antibody were then determined with the horseradish peroxidase substrate o-phenylenediamine and quantified by measuring absorbance at 490 nm. The binding of antibodies served as a measure of virus density on the solid phase.

FIG. 4

Locations of HA mutations in NA deletion variants. Mutated amino acids are represented by the filled residues, modeled on the A/Aichi/68 H3 HA structure (32). For clarity, only the head portion of HA1 is displayed. Sialic acid in the receptor-binding pocket is represented as a black ball-and-stick model. Mutations at residues 135 and 145, found at the right side of the receptor-binding site, are common to all viruses with greatly reduced affinity for viral receptors. This figure was generated with RasMol software (http://www.umass.edu/microbio/rasmol).