Hemagglutinin Specificity and Neuraminidase Coding Capacity of Neuraminidase-Deficient Influenza Viruses

Abstract

Neuraminidase (NA)-deficient mutant virus stocks have been obtained by passaging A/NWS/33HA-tern/Australia/G70c/75NA (H1N9) influenza virus in medium containing neuraminidase from Micromonospora viridifaciens and antiserum against the influenza NA. Growth of the resulting mutants is dependent on addition of bacterial neuraminidase to the medium. Nucleotide sequence analysis showed large single deletions in the NA genes, with both ends of the NA gene segments conserved. These RNA fragments all have the capacity to code for a peptide that contains the N-terminal "tail" and membrane-anchoring region of the NA, but the presence of this peptide has not been demonstrated in virions or infected cells. In contrast to the ease of selection of NA-deficient mutants from the H1N9 virus, no mutants were selected from three other viruses. The HA-coding segments of parental H1N9 and mutant NWSc-Mvi predict a change of Pro to His at residue 227 (H3 numbering), close to the receptor-binding site of H3 HA, compared to the HA of an H1N2 reassortant that contains the NWS/33 HA gene. This change may contribute to an altered HA specificity that allows selection of mutants that can infect cells in the presence of high levels of NA activity. It appears that the role of NA in influenza infection is to remove sialic acid from the HA rather than to destroy receptors on cells.

Influenza A Viruses Lacking Sialidase Activity Can Undergo Multiple Cycles of Replication in Cell Culture, Eggs, or Mice

“…As with the NWS-MviA virus (33), the CK2-29 virus, after extensive passage in tissue culture, eggs, or mice, retained a truncated NA gene with the capacity to direct synthesis of the cytoplasmic tail and some (our mutants) or all (NWS-MviA virus [33]) of the transmembrane domain and a short stalk. These findings suggest that the truncated NA protein may be important in the virus replication cycle, perhaps in virion morphogenesis and stability.…”

“…The resultant NWS-MviA virus contains an internal truncation of a large portion of the NA gene (bases 140 to 1248), so that the coding region generates the cytoplasmic and transmembrane regions of the protein as well as a small portion of the stalk (33). The virus therefore lacks sialidase activity, resulting in aggregation of NWS-MviA progeny virions at the host cell surface (16).…”

Genome packaging in influenza A virus

“…Influenza A genome packaging deficient influenza viruses could be selected for in tissue culture using exogenously supplied bacterial neuraminidase and antibodies against the viral NA (Liu & Air, 1993;Yang et al, 1997). These viruses all retained internally deleted versions of segment 6, structurally akin to DI RNAs, strongly suggesting a positive selection pressure to retain the terminal regions of the vRNA (Yang et al, 1997).…”

“…These viruses all retained internally deleted versions of segment 6, structurally akin to DI RNAs, strongly suggesting a positive selection pressure to retain the terminal regions of the vRNA (Yang et al, 1997). At the time, it could not be determined whether this reflected a need for the truncated vRNAs and/or the short NA peptides they encoded.…”

Adaptation of Influenza A Viruses to Cells Expressing Low Levels of Sialic Acid Leads to Loss of Neuraminidase Activity

“…In previous studies, the passage of influenza A viruses in the presence of an exogenous bacterial sialidase activity and antibodies to the viral NA led to deletion of the viral NA gene (14,15,29). Moreover, NA mutants obtained by such passaging were able to grow in cell cultures lacking exogenous sialidase activity, as well as in eggs and mice, as a result of compensatory mutations in the HA protein that reduce the molecule's affinity for sialic acid residues (8).…”