BELGIAN SOCIETY FOR MICROBIOLOGY - Cell Signaling in Host-Microbe Interactions location:Brussels, Belgium date:18 November 2014
Introduction: The influenza virus hemagglutinin (HA) plays a crucial role in fusion between the viral and endosomal membranes during influenza virus entry. This fusion process can be blocked by compounds interfering with the acid-induced conformational change of HA.
Methods: After identification of the anti-influenza virus activity of CIM07 in MDCK cells, its mechanism of action was unraveled by performing time-of-addition studies, entry assays using confocal microscopy and HA-mediated fusion (i.e. syncytium formation and tryptic digestion) assays. Finally, CIM07-resistant mutants were selected and characterized.
Results: In Madin-Darby canine kidney (MDCK) cells infected with influenza virus A/H3N2, the 50% effective concentration of CIM07 was 0.20 µM, as determined by microscopy of the viral cytopathic effect and MTS cell viability assay. The concentration causing minimal alterations in cell morphology was ≥25 µM. Similar activity was seen for other A/H3N2 strains, but no activity was noted for influenza A/H1N1 and B viruses. In time-of-addition studies, CIM07 lost activity when added 1 hour or later post infection, showing that CIM07 inhibits an early step in virus replication. CIM07 was proven to completely prevent the nuclear entry of influenza virus, based on confocal microscopy with anti-nucleoprotein staining. The inhibitory effect of CIM07 on low pH-induced syncytium formation in HA-expressing cells indicated that CIM07 blocks HA-mediated membrane fusion. Definite proof for this mode of action was provided by the tryptic digestion assay, in which CIM07 prevented the acid-induced conversion of HA to its trypsin-sensitive conformation. CIM07-resistant mutants, selected after three passages in 0.37-10 µM CIM07, were plaque-purified. Phenotypic and genotypic characterization of these resistant viruses is currently ongoing.
Discussion: CIM07 represents an excellent tool to further delineate the structural changes during the low pH-induced HA refolding process. The resistance mutations allow to identify its binding pocket and design new CIM07 derivatives with improved activity.