Enveloped viruses use use spike proteins as molecular mimics of host molecules in order to bind to target cell receptors and gain entry into cells. However, these spikes serve as convenient antigenic surfaces for immune system recognition. Mammalian viruses thus face tremendous selective pressures to change their molecular profiles to evade astoundingly responsive immune systems capable of recognizing and destroying viral particles and infected cells. In many cases, natural selection continually yields viral strains that vary considerably in the antigenic regions of spike proteins. These genetic variants may arise and spread through target species periodically, as in the case of annual human flu virus infections. Or, they may be produced during the course of a single infection, as in the HIV variants that arise in the large number of replication cycles that occur over years within a human individual. This tutorial concerns the structure/function/variability of the hemagglutinin spike protein of the human influenza virus.
Hemagglutinin, displayed at left, is one of two virally-coded integral envelope proteins of the influenza virus. Hemagglutinin is responsible for host cell binding and subsequent fusion of viral and host membranes in the endosome after the virus has been taken up by endocytosis. In the first step of infection it binds to sialic acid residues of glycosylated receptor proteins on target cell surfaces. After endocytosis brings the virus into the cell in an endosome, acidification of the endosome induces conformational changes in hemagglutinin that promote fusion with the endosomal membrane, thereby promoting the release of the flu virus into the host cytoplasm.
The elongate hemagglutinin protein is a trimer that measures ~135 Å from insertion in the envelope membrane to its tip.
Each of the three subunits comprises two chains produced by proteolytic cleavage of a monomeric precursor protein. The six resulting chains are three HA1s and three HA2s.
Let's examine one monomer of hemagglutinin. The HA1 subunit (328 residues) is an elongate structure reaching from the N-terminus at the viral membrane end of the molecule along the stem of the subunit before forming a globular tip. HA1 then returns part way down the stem of the monomer, ending at the C-terminus.
Part of the distal end of HA1 forms an 8-stranded antiparallel β-sheet motif termed a "jelly roll." A short α-helix forms in the loop that separates strands 3 and 4 of the jelly roll.
Residues from one side of this α-helix and from residues near the top of the jelly roll form a pocket that is the sialic acid binding site for each monomer of the hemagglutinin trimer.
A remarkable feature of the HA2 subunit (221 residues) is the two antiparallel α-helices that form part of the stem of the molecule. One of these is among the longest α-helices known in globular proteins (~ 75Å).
The HA1 and HA2 chains of each monomer are connected via a single disulfide bond. HA2 terminates in an α-helical structure near the protease cleavage site.
Stabilization of the hemagglutinin trimer arises from interactions between three major HA2α-helices in the formation of a triple-stranded coiled coil in the interior of the trimer. The N-terminal (top) half of the coiled-coil superhelix is tightly packed with several nonpolar residues in van der Waals contact around the 3-fold axis. The C-terminus end of the superhelix expands away from the axis with polar and charged residues from each monomer experiencing electrostatic repulsion from like residues in the other monomers.
A soluble fragment of hemagglutinin at low pH has been isolated and characterized (Bullough, et al., 1994). This fragment (TBHA2) is prepared at pH 5.0 by digestion with trypsin and thermolysin and contains the first 27 residues of HA1 and residues 38-175 of HA2. Although many of the original hemagglutinin residues are lost in this digestion, the major conformational change caused by the acidic environment in the endosome is clear when one compares the conformation of the original HA2 subunit (BHA-left) to that of TBHA2 (right). The subunits are colored in rainbow from amino to carboxy. Residues 55-76 in BHA are recruited to an α-helix in TBHA2 which extends the α-helix of residues 40-55 in BHA 100 Å towards the endosome membrane in TBHA2 (endosome membrane is up). The α-helix of BHA is also moved slightly away from the viral membrane in TBHA2 and the β-sheet/α-helix structure of BHA follows towards the endosome membrane. The functional consequence of the endosomal refolding is a translocation of residues at the end of the α-helix (not shown) to the endosome membrane, where they fuse with it. The resulting α-helix (110 Å) in TBHA2 is one of the longest known in any protein.
A simulation of the folding changes induced by endosomal acidification can be viewed. Although the starting and ending structures are genuine models based on crystallographic data, the intermediate transitions are based on linear interpolation and some energy minimization and are only possible structures. As above, note the projection of the fusion α-helix towards the endosomal membrane (up). This simulation was generated using the Yale Morph Server at the Database of Macromolecular Movements, maintained by the Gerstein lab.
The mechanism of pH-induced conformational change of flu HA is of considerable interest. Kampmann, et al. (2006) provide evidence that protenation of specific histidine residues, highly conserved among influenza serotypes, is involved in this process. Histidine side chains, with a pKa in the pH range of ~6-7, are singly protenated at pH 7 and are neutral. In this state they are capable of serving as hydrogen bond acceptors, interacting strongly with positively charged residues in their environment in the prefusion conformer. Upon acidification of the endosomal vesicle, these histidine side chains have the potential to become doubly protenated and therefore positively charged. This protenation can cause the repulsion from positively charged residues in the prefusion environment, can disrupt initial hydrogen bonding of histidine side chain atoms, and can promote the formation of new H-bonds and salt bridges with negatively charged residues in the postfusion environment. These pH-induced changes in bonding potential likely play a key role in the generation of the "fusion active" conformer of HA. As examples, consider the disposition of two key histidines in the pre- and post-fusion forms of HA.
Four major antigenic sites have been located on the hemagglutinin monomer. Site A is a loop that protrudes 8 Å distally from the molecular surface. Site B combines external residues of an α-helix with several residues of the pocket responsible for sialic acid binding. Site C is a bulge 60 Å from the distal tip of the molecule. Site D is located in two of the β-sheets of the jelly roll (see Structure, above). Evidence suggests that single amino acid substitutions within these four regions result in the ability of flu virus to escape immune surveillance and to spread worldwide every year. In addition to these minor changes, major changes in the antigenic regions have produced the extremely virulent strains that caused the lethal flu pandemics of 1957 and 1968.
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