Inhibition of influenza A virus replication by compounds interfering with the fusogenic function of the viral hemagglutinin

Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds i nhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting th at an uncoating step was blocked. Sequencing of hemagglutinin (IIA) genes o f several independent mutant viruses resistant to the compounds revealed si ngle amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compou nd in inhibiting virus replication was synthesized. It was also more effect ive in blocking other manifestations of the low-pH-induced conformational c hange in HA including virus inactivation, virus-induced hemolysis of erythr ocytes, and susceptibility of the HA to proteolytic degradation. Both compo unds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type v irus. The possible functional relationship between M2 and IIA suggested by these results is discussed.