Influenza Virus Hemagglutinin Regulates Membrane Fusion By Fine-Tuning Of Its Histidines

The influenza virus hemagglutinin (HA) is known to play an essential role in virus infection. It is not only involved in cell receptor binding and uptake by endocytosis, but moreover, after internalization and trafficking to early and late endosomes, induces membrane fusion which leads to the release of the viral genome into the cell. It has been shown for several influenza strains that the pH of fusion of HA can be shifted by modulating its stability which results in altered infectivity of the virus. However, the underlying molecular mechanisms that lead to an altered pH dependence of membrane fusion are still unknown. Using experimental approaches and Molecular Dynamics studies we demonstrate that the introduction of charges in the vicinity of certain His residues can change its protonation state, thereby influencing the pH dependant conformational change of the HA protein. Histidines at key structural locations were identified and neighboring residues were mutated in the HA of highly (HP) or low pathogenic (LP) H5N1 and X31. Fusion activity and stability of recombinant proteins and resulting virus particles were assessed by fluorescence dequenching. First results showed that charges, especially at position 216, in the vicinity of His184 have a significant effect on the stability of the HA1 ectodomain in both H3 and H5 HA. Interestingly, Arginine at position 216 is naturally present in the H5 HP whereas the H5 LP has a Glutamate at this position. We could show that the exchange of these charges in both variants rescues the pH dependence of fusion of HP and LP, respectively. We suggest that influenza viruses adjust their pH of activation by the exchange of charged residues in the vicinity of conserved Histidines for optimized spread and stability.