Characterization of the Carbohydrate-Binding Agents HHA, GNA, and UDA as Inhibitors of Influenza A and B Virus Replication.

Here, we report on the anti-influenza virus activity of the mannose-binding agents hybrid agglutinin (HHA) and agglutinin (GNA) and the (-acetylglucosamine) -specific agglutinin (UDA). These carbohydrate-binding agents (CBA) strongly inhibited various influenza A(H1N1), A(H3N2), and B viruses , with 50% effective concentration values ranging from 0.016 to 83 nM, generating selectivity indexes up to 125,000. Somewhat less activity was observed against A/Puerto Rico/8/34 and an A(H1N1)pdm09 strain. In time-of-addition experiments, these CBA lost their inhibitory activity when added 30 min postinfection (p.i.). Interference with virus entry processes was also evident from strong inhibition of virus-induced hemolysis at low pH. However, a direct effect on acid-induced refolding of the viral hemagglutinin (HA) was excluded by the tryptic digestion assay. Instead, HHA treatment of HA-expressing cells led to a significant reduction of plasma membrane mobility. Crosslinking of membrane glycoproteins, through interaction with HA, could also explain the inhibitory effect on the release of newly formed virions when HHA was added at 6 h p.i. These CBA presumably interact with one or more -glycans on the globular head of HA, since their absence led to reduced activity against mutant influenza B viruses and HHA-resistant A(H1N1) viruses. The latter condition emerged only after 33 cell culture passages in the continuous presence of HHA, and the A(H3N2) virus retained full sensitivity even after 50 passages. Thus, these CBA qualify as potent inhibitors of influenza A and B viruses with a pleiotropic mechanism of action and a high barrier for viral resistance.