Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus

Abstract

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.

FIG. 1

(A) Nucleic acid sequences and the locations of inserts in the NAs of five SD0 egg-adapted strains. The proposed transmembrane and catalytic head domains of the NA are indicated. Inserted sequences are identical to the PB1, PB2, and NP gene segments of WSN virus. The 21EA strain contains NP nucleotides 523 to 586; 18EA contains PB2 nucleotides 928 to 981; 10EA contains PB1 nucleotides 1814 to 1851; 7EA contains PB1 nucleotides 2063 to 2092; and 0EA contains PB2 nucleotides 720 to 784. Bold italic sequences in 21EA indicate the NP-specific primer (primer NPinsert) synthesized for PCR detection (see Table 2). (B) Deduced amino acid sequence and locations of inserted amino acids in five SD0 egg-adapted strains.

FIG. 1

(A) Nucleic acid sequences and the locations of inserts in the NAs of five SD0 egg-adapted strains. The proposed transmembrane and catalytic head domains of the NA are indicated. Inserted sequences are identical to the PB1, PB2, and NP gene segments of WSN virus. The 21EA strain contains NP nucleotides 523 to 586; 18EA contains PB2 nucleotides 928 to 981; 10EA contains PB1 nucleotides 1814 to 1851; 7EA contains PB1 nucleotides 2063 to 2092; and 0EA contains PB2 nucleotides 720 to 784. Bold italic sequences in 21EA indicate the NP-specific primer (primer NPinsert) synthesized for PCR detection (see Table 2). (B) Deduced amino acid sequence and locations of inserted amino acids in five SD0 egg-adapted strains.

FIG. 2

Three-dimensional structure of the HA monomer (30), indicating the location of each HA mutation discovered in three SD0 egg-adapted strains, relative to sialic acid (Neu5Ac) binding.