INHIBITION OF INFLUENZA-VIRUS HEMAGGLUTININ-MEDIATED MEMBRANE-FUSION BY A COMPOUND RELATED TO PODOCARPIC ACID

Entry of influenza virus into the host cell is dependent on the fusion of the viral envelope with the endosomal membrane and is mediated by a low-pH-induced change of the viral hemagglutinin (HA) to a conformat ion that is fusogenic. A compound related to podocarpic acid (180299] was identified that inhibits multicycle replication of influenza A/Kaw asaki/86 (H1N1)Virus in culture. Treatment of Madin-Darby canine kidne y (MDCK) cells with 180299 at 1 h before infection resulted in the inh ibition of viral protein synthesis. Addition of 20 mu g of 180299/ml a t 1 h p.i. had no effect, indicating that 180299 affects an early step of the influenza viral replication cycle. Genetic analysis of reassor tants between sensitive and resistant viruses demonstrated that hemagg lutinin (HA) conferred the 180299-resistant (180299(r)) phenotype. Twe lve independent isolates of influenza A/Kawasaki/86 were selected for resistance to 180299, and sequence analysis revealed that each of thes e viruses contained amino acid substitutions in the HA. These mutation s are dispersed throughout the HA primary amino acid sequence and clus ter in one of two regions: the interface between HA(1) and HA(2) and i n a region near the fusion domain of HA(2). When compared with the par ent virus, the pH-of-inactivation of the resistant mutants was increas ed by 0.3 to 0.6 pH unit, suggesting that the mutant HAs undergo the c onformational change at an elevated pH. Fusion of human erythrocytes t o MDCK cells infected with parent influenza A/Kawasaki/86 was inhibite d by 180299 (0.1-10 mu g/ml) in a concentration-dependent: manner, whe reas fusion of erythrocytes to MDCK cells infected with 180299(r) muta nts was not: affected. These results suggest that 180299 interacts wit h the neutral pH conformation of influenza A HA and prevents the low-p H-induced change of HA to its fusogenic conformation, (C) 1998 Academi c Press.