Modulation of the pH Stability of Influenza Virus Hemagglutinin: A Host Cell Adaptation Strategy.

Proteins undergo dynamic structural changes to function within the range of physical and chemical conditions of their microenvironments. Changes in these environments affect their activity unless the respective mutations preserve their proper function. Here, we examine the influenza A virus spike protein hemagglutinin (HA), which undergoes a dynamic conformational change that is essential to the viral life cycle and is dependent on endosomal pH. Since the cells of different potential hosts exhibit different levels of pH, the virus can only cross species barriers if HA undergoes mutations that still permit the structural change to occur. This key event occurs after influenza A enters the host cell via the endocytic route, during its intracellular transport inside endosomes. The acidic pH inside these vesicles triggers a major structural transition of HA that induces fusion of the viral envelope and the endosomal membrane, and permits the release of the viral genome. HA experiences specific mutations that alter its pH stability and allow the conformational changes required for fusion in different hosts, despite the differences in the degree of acidification of their endosomes. Experimental and theoretical studies over the past few years have provided detailed insights into the structural aspects of the mutational changes that alter its susceptibility to different pH thresholds. We will illustrate how such mutations modify the protein's structure and consequently its pH stability. These changes make HA an excellent model of the way subtle structural modifications affect a protein's stability and enable it to function in diverse environments.

A histidine residue of the influenza virus hemagglutinin controls the pH dependence of the conformational change mediating membrane fusion.

UNLABELLED: The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKas match the pHs of late endosomes (pK(a) of ∼ 6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines with different amino acids and analyzing the effect of these mutations on conformational change and fusion, we found that His184, but not His110, plays an essential role in the pH dependence of the conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central in a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in the vicinity of histidine may affect its pK(a). The HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly pathogenic as well as the low pathogenic H5 HA, we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pK(a)-modulating effect of neighboring residues. IMPORTANCE: The main pathogenic determinant of influenza viruses, the hemagglutinin (HA) protein, triggers a key step of the infection process: the fusion of the virus envelope with the endosomal membrane releasing the viral genome. Whereas essential aspects of the fusion-inducing mechanism of HA at low pH are well understood, the molecular trigger of the pH-dependent conformational change inducing fusion has been unclear. We provide evidence that His184 regulates the pH dependence of the HA conformational change via its pK(a). Mutations of neighboring residues which may affect the pK(a) of His184 could play an important role in virus adaptation to a specific host. We suggest that mutation of neighboring residue 216, which is present in all highly pathogenic phenotypes of H5N1 influenza virus strains, contributed to the adaptation of these viruses to the human host via its effect on the pKa of His184.

Receptor binding and pH stability - how influenza A virus hemagglutinin affects host-specific virus infection.

Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus. Adaptation to different hosts in vitro was shown to require mutations within HA altering the receptor binding and/or fusion behavior of the respective virus strain. Human adapted influenza virus strains (H1N1, H3N2, H2N2) as well as recent avian influenza virus strains (H5, H7 and H9 subtypes) which gained the ability to infect humans mostly contained mutations in the receptor binding site (RBS) of HA enabling increased binding affinity of these viruses to human type (α-2,6 linked sialic acid) receptors. Thus, the receptor binding specificity seems to be the major requirement for successful adaptation to the human host; however, the RBS is not the only determinant of host specificity. Increased binding to a certain cell type does not always correlate with infection efficiency. Furthermore, viruses carrying mutations in the RBS often resulted in reduced viral fitness and were still unable to transmit between mammals. Recently, the pH stability of HA was reported to affect the transmissibility of influenza viruses. This review summarizes recent findings on the adaptation of influenza A viruses to the human host and related amino acid substitutions resulting in altered receptor binding specificity and/or modulated fusion pH of HA. Furthermore, the role of these properties (receptor specificity and pH stability of HA) for adaptation to and transmissibility in the human host is discussed. This article is part of a Special Issue entitled: Viral Membrane Proteins -- Channels for Cellular Networking.