The initial characterization of the newly isolated ZM1 variant revealed that it harbors two HN mutations (20
). One of the mutations, T193I, is located at the same residue as the single HN mutation, T193A, in the C0 variant. Since C0 failed to show the strong 4-GU-DANA resistance characteristics of ZM1 (20
), it seemed that these differences might be due to either the specific substitution at position 193 or to the I567V mutation in ZM1 HN. This latter possibility was now disproved by the use of singly mutated HNs: we obtained conclusive evidence that the deviations (from WT HN) in all the examined properties of ZM1 variant HN are due to T193I.
The structural similarities between influenza virus NA and paramyxovirus HN suggest that homologous residues might serve similar functional roles. The HPF3 residue T193, which is altered in our ZM1 and C0 variants (to an I and an A, respectively), corresponds to residue E119 in the active-site pocket of influenza virus NA (25
) and to NDV HN residue I175 (2
). Crystallographic studies of NDV HN indicate that, as in influenza virus NA, the residue at this position lies within the sialic acid binding pocket (3
). Substituting residues at I175 in NDV was shown to diminish NA activity, to prevent receptor binding in HAD assays, and to affect fusion promotion ability, clearly indicating the functional importance of this residue for the protein (3
Due to the structural parallels with influenza virus NA and NDV HN, it seemed likely that HPF3 HN residue T193 provides important interactions with sialic acid receptors and that a mutation at this site could confer resistance to 4-GU-DANA. It was somewhat surprising that the C0 T193A alteration in HPF3 HN did not seem to confer 4-GU-DANA resistance in terms of infectivity or HAD on C0-infected cells (20
). We therefore reexamined this question by using C0 HN-expressing cells and the more sensitive assays for HN-receptor interaction developed in the present study. The results showed that T193A did confer significant 4-GU-DANA resistance to the C0 variant HN relative to the WT HN, further emphasizing the importance of residue 193. In addition, the less pronounced 4-GU-DANA resistance of C0 HN (T193A) relative to ZM1 HN (T193I) indicates that the degree of functional alterations depends strongly on whether the original amino acid at this site was replaced by A or I.
ZM1 is the first HPF3 variant found to exhibit increased NA activity. The association of this increase with a rise in the receptor binding avidity of ZM1 HN is in accord with our previous suggestion that a balance between HN′s NA activity and its receptor binding activity is critical for HPF3 propagation (22
). A manifestation of such a balance can be seen in experiments on the release of bound RBC from HN-expressing cells at 37°C. For cells expressing ZM1 variant HN, with its increased receptor binding activity as well as NA activity, RBC release was as fast as it was for WT HN-expressing cells (Fig. ). In variants C22 and C0, studied in parallel with ZM1, no change in NA activity accompanied the elevation of receptor binding avidity (19
); consequently, C22 or C0 HN-expressing cells were much less capable of releasing RBC than were ZM1 (or WT) HN-expressing cells, even though the elevation of their receptor binding avidity was less (C22) or much less (C0) pronounced than that of ZM1 HN.
This gradation in the properties of these 4-GU-DANA-resistant variants provided insight into the nature of their resistance. Since C22 and C0 exhibited resistant behavior despite having normal (i.e., WT) NA activity, it seems likely that the 4-GU-DANA resistance of ZM1 is not a result of its increased NA activity. In contrast, the increase in the receptor binding avidity of the variant HNs was directly related to the extent of their 4-GU-DANA resistance, suggesting that this resistance is a consequence of increased receptor binding avidity. Elucidation of the underlying molecular mechanism will require characterization of the site at which 4-GU-DANA binds to HPF3 HN and determination of the affinity of 4-GU-DANA to the different HNs.
In influenza viruses, HA mediates receptor attachment as well as fusion, while the second envelope protein, NA, provides the receptor cleaving activity that permits the release of newly budded virions. In the presence of 4-GU-DANA, which is a powerful selective inhibitor of influenza virus NA (25
), these progeny virions remain aggregated on the cell surface, with the consequent curtailment of the spread of infection. In the case of HPF3, on the other hand, 4-GU-DANA hinders infectivity in vitro by interfering with HN-receptor interaction and thus precluding the subsequent steps of fusion and viral entry (6
). If infection does occur, the release of progeny HPF3 virions is not hindered by 4-GU-DANA, even though it inhibits the NA activity of HN. Rather, by inhibiting HN-receptor binding, 4-GU-DANA prevents virion aggregation itself, thus obviating the need for receptor cleavage by the NA activity of HN.
The different mechanisms whereby 4-GU-DANA interferes with influenza virus and HPF3 infectivities in vitro suggested that the type of variants arising under the selective pressure of 4-GU-DANA would be different for these two viruses. It has been shown that prolonged growth of influenza viruses in the presence of 4-GU-DANA gives rise to variants with a mutation in the active site of NA but that variants that emerge first are those harboring HA mutations in the receptor binding activity (17
). A partial loss of receptor binding ability would diminish the aggregation of progeny virions, and the consequent availability of free virions to infect more cells would counterbalance the effect of 4-GU-DANA, which impedes influenza virus infectivity by preventing the release of progeny virions. Thus, influenza virus escape variants were expected, and found, to include HA mutants with decreased receptor binding avidity. For HPF3, where 4-GU-DANA curtails infectivity by inhibiting HN-receptor interaction, it is reasonable to hypothesize that HN mutants with increased receptor binding avidity are those that can escape 4-GU-DANA's growth inhibitory effect. The present results are in accord with this hypothesis.