Hemagglutinin

The most striking feature of influenzavirus is the layer of spikes projecting outward from the surface (Fig. I). These spikes are the hemagglutinin (FIA) proteins of which there are estimated to be a few hundred copies per virion (Ruigrok et al, 1984 Amano and Hosaka, 1992). The HA spikes carry out both receptor binding and membrane fusion during infection. ELA of influenza A virus was the first enveloped virus surface protein to be studied by X-ray crystallography (Wilson et al, 1981), 

Influenza HA, in the form of HAq, begins folding as a single polypeptide chain. It presumably folds into the thermodynamically most stable, kinetically accessible state, although this supposition has not been proven. 

Peptide Bond Cleavage and Rearrangement to the Native State of the 

For many viruses, a posttranslational peptide bond cleavage at a particular site in an envelope protein is required to prime the virus surface for its membrane fusion activity. Influenza HAq is thus cleaved 

Comparison of the HAq structure (Chen et al, 1998) with the structure of HA1-HA2 (Wilson et al, 1981) reveals that, on cleavage, the new HAi carboxy terminus and the HA2 amino terminus are displaced relative to one another by 22 A. The most significant change is that the newly liberated amino terminus of HA2 dives into a pocket near the base of the coiled-coil stalk (Fig. 4). Cleavage of HAq is required for this alternate packing because there is no room for a polypeptide chain to both enter 

As mentioned in Section 17.2.1, HA is a spike glycoprotein anchored to the virus lipid membrane [50], This glycoprotein functions as a receptor-binding protein and is responsible for the first step of viral infection when it binds to sialic acid residues of receptor glycoproteins on host cells [18]. When the virus is endocytosed into the cell, the low pH (5-6) changes the structure of HA, and this new fusion-active state triggers the fusion of the viral membrane and the endosome membrane, ultimately allowing entry of the viral nucleocapsid into the cytosol of the host cell [18]. 

HA (Fig. 17.5 [51]) is a trimer and measures approximately 135 A from insertion in the envelope membrane to its tip [19]. The three sites where sialic acid should bind are approximately 40A apart from each other [19, 52], 

FIGURE 17.4 Representation of the three major sialic acid subfamilies. 

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Arachin, the counterpart of glycinin in peanuts, consists of subunits of 60,000—70,000 mol wt which on reduction with 2-mercaptoethanol yield polypeptides of 41,000—48,000 and 21,000 mol wt (17) analogous to the behavior of glycinin. In addition to the storage proteins, oilseeds contain a variety of minor proteins, including trypsin inhibitors, hemagglutinins, and enzymes. Examples of the last are urease and Hpoxygenase in soybeans. 

Standardization and Testing". The final vaccine is tested for safety, potency, and residual chemicals. Safety includes testing for endotoxin and stetihty. Potency is evaluated by quantitative determination of the amount of hemagglutinin in the vaccine. Antibody to this glycoprotein is associated with protection. The single radial immunodiffusion (SKID) technique is used to standardi2e the mass of this protein in comparison to a reference preparation. 

The jelly roll barrel is thus conceptually simple, but it can be quite puzzling if it is not considered in this way. Discussion of these structures will be exemplified in this chapter by hemagglutinin and in Chapter 16 by viral coat proteins. 

Figure 5.19 Schematic picture of a single subunit of influenza virus hemagglutinin. The two polypeptide chains HAj and HA2 are held together by disulfide bridges.

We have already discussed one envelope protein of influenza virus, neuraminidase, as an example of an up-and-down antiparallel p motif. In the second envelope protein, hemagglutinin, one domain of the polypeptide chain is folded into a jelly roll motif. We shall now look at some other features of hemagglutinin that are important for its biological function. 

Progeny vims particles then bud from patches of the infected cell s plasma membrane that contain both the viral hemagglutinin and neuraminidase. The viral envelopes therefore contain both viral membrane proteins but no cellular membrane proteins. 

The hemagglutinin trimer molecule is 135 A long (from membrane to tip) and varies in cross-section between 15 A and 40 A. It is thus an unusually... 

The binding site is located at the tip of the subunit within the jelly roll structure (Figure 5.23). The sialic acid moiety of the hemagglutinin inhibitors binds in the center of a broad pocket on the surface of the barrel (Figure 5.24). In addition to this groove there is a hydrophobic channel that can accomodate large hydrophobic substituents at the C2 position of sialic acid (Figures 5.22 and 5.24). 

In addition to binding to sialic acid residues of the carbohydrate side chains of cellular proteins that the virus exploits as receptors, hemagglutinin has a second function in the infection of host cells. Viruses, bound to the plasma membrane via their membrane receptors, are taken into the cells by endocytosis. Proton pumps in the membrane of endocytic vesicles that now contain the bound viruses cause an accumulation of protons and a consequent lowering of the pH inside the vesicles. The acidic pH (below pH 6) allows hemagglutinin to fulfill its second role, namely, to act as a membrane fusogen by inducing the fusion of the viral envelope membrane with the membrane of the endosome. This expels the viral RNA into the cytoplasm, where it can begin to replicate. 

This fusogenic activity of influenza hemagglutinin is frequently exploited in the laboratory. If, for example, the virus is bound to cells at a temperature too low for endocytosis and then the pH of the external medium is lowered, the hemagglutinin causes direct fusion of the viral envelope with the plasma membrane infection is achieved without endocytosis. Similarly, artificial vesicles with hemagglutinin in their membrane and other molecules in their lumen can be caused to fuse with cells by first allowing the vesicles to bind to the plasma membrane via the hemagglutinin and then lowering the pH of the medium. In this way the contents of the vesicles are delivered to the recipient cell s cytoplasm. 

Figure 5.23 The globular head of the hemagglutinin subunit Is a distorted jelly roll stmcture (a). P strand 1 contains a long Insertion, and P strand 8 contains a bulge in the corresponding position. Each of these two strands is therefore subdivided Into shorter P strands. The loop region between P strands 3 and 4 contains a short a helix, which forms one side of the receptor binding site (yellow circle). A schematic diagram (b) Illustrates the organization of the p strands into a jelly roll motif.

Figure 5.24 Space-filling model (green) of the sialic acid binding domain of hemagglutinin with a bound inhibitor (red) Illustrating the different binding grooves. The sialic acid moiety of the Inhibitor binds in the central groove. A large hydrophobic substituent, Ri, at the Cz position of sialic acid binds in a hydrophobic channel that runs from the central groove to the bottom of the domain. (Adapted from S.J. Watowich et al.. Structure 2 719-731, 1994.)...

Figure 5.27 Schematic representation of a model for the conformational change of hemagglutinin that at low pH brings the fusion peptide to the same end of the molecule as the receptor binding site. The fusion peptide (purple) is at the end of heUx A about 100 A away from the receptor binding site in the high pH form. In the low pH fragment this region of helix A has moved about 100 A towards the area where the receptor binding sites are expected to be in the intact hemagglutinin molecule. (Adapted from D. Stuart, Nature 371 19-20, 1994.)...

Daniels, R.S., et al. Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell 40 431-439, 1985. 

Wiley, D.C., Skehel, JJ. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 56 365-394, 1987. 

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