Hemagglutinin esterase (HEs) is a glycoprotein that certain enveloped viruses possess and use as an invading mechanism. HEs helps in the attachment and destruction of certain sialic acid receptors that are found on the host cell surface. Viruses that possess HEs include influenza C virus, toroviruses, and coronaviruses of the subgenus Embecovirus (which does not include SARS-like coronaviruses). HEs is a dimer transmembrane protein consisting of two monomers, each monomer is made of three domains. The three domains are: membrane fusion, esterase, and receptor binding domains.
The different HEs enzyme activities include: receptor binding activity, receptor hydrolysis (esterase) activity, and membrane fusion activity. The receptor binding activity involve the attachment of HEs to N-acetyl-9-O-acetylneuraminic acid (9-O-Ac- Neu5Ac) of glycolipids and glycoproteins and in turn serve as viral receptor. Receptor hydrolysis (esterase) activity allows virus particles to escape the infected cell by removing an acetyl group from the C9 position of terminal 9-O-Ac-Neu5Ac residues. Membrane fusion activity helps in incorporation viral genome into the host cell cytoplasm by enhancing the attachment between the viral envelope and host cell membrane.
In certain influenza viruses, the cell surface consists of both hemagglutinin (HA) and neuraminidase (NA) proteins that encompass enzymatic activities, whereas hemagglutinin-esterase fusion (HEF) proteins have been found to be the primary single spike protein that combines all of the enzymatic activities listed above. HEF proteins have been tested to be high-temperature and low-pH resistant and are the primary source of virulence in viruses. Influenza C have been shown to have unique HEF structure proteins that enhance its ability to infect the host cell compared to influenza A and B.
The folding of different domains in the hemagglutinin-esterase protein is important for intracellular transport of proteins from the endoplasmic reticulum to the Golgi apparatus. The presence of oligosaccharide chains in the E, F, and R domains of the HE enzyme also influence intracellular transport. Acylation of the hemagglutinin-esterase has shown to play an essential role in virus particle assembly replication. The exact process of enzyme catalytic cleavage has not yet been detailed out. However, proteolytic cleavage must occur before hemagglutinin-esterase membrane fusion activity. HEF proteins have a unique spikes hexagonal arrangement. This feature is unique to influenza C virus particles. The arrangement is a covering outside of the particle.
Structure Certain studies revealed that coronavirus and toroviruses HE was originated from HEF glycoprotein that is found in influenza C viruses which resulted from alteration of hemagglutinin esterase from a trimer into a dimer glycoprotein. During this process, the receptor destroying enzyme acetyl esterase domain stayed unchanged. However, the HE receptor binding domain has been altered in which that the ligand is bound in opposite orientation than before. Both coronavirus and toroviruses HE monomers are made up of the same three domains: central esterase/hydrolase domain, receptor binding lectin domain, and membrane proximal domain which is small. The two monomers of HE dimer in both CoV and ToV involve the same two contact regions (CR 1 and 2). CR 1 contain the receptor binding domain and contact region 2 that contain membrane proximal domain. Yet, ToV HE contacts region 2 contain additional esterase domain. As a result, the CR 2 surface is larger in ToV HEs than in CoV HEs. However, close to the carboxylic terminal membrane anchor, there are number of disulfide bridges between Cys385 of coronavirus HE that in turn keep the HE dimers connected to each other.
In CoV HE, the two R domain beta sheets are connected to each other forming a continuous intermolecular beta sheet across the dimer interface. On the other hand, in ToV they are oriented at angles. As a result, the beta sheet of receptor binding domain in ToV is more twisted, the contact region 1 is smaller, and the R domains position are shifted along the Beta strands compared to CoV.
Crystalline structure "Initial studies using electron microscopy showed that the HEF spike forms a mushroom-shaped trimer consisting of a membrane-near stalk and a globular head".
Later studies were able to examine and show a higher resolution structure (4.5 Å) of the hemagglutinin esterase fusion trimer using X-ray crystallography of the bromelain-cleaved ectodomain. Both hemagglutinin and hemagglutinin esterase fusion protein are similar in terms of structure and the folding of individual segments. yet, only 12% amino acid are identical between HA and HEF. One significant difference between HE and HEF is the presence of an additional bulge in HEF globular domain (bottom part of the domain) which contains the esterase region. The receptor-binding region in both HA and HEF is found in the upper part of the domain and contain only HEF1 residues. The stalk is made of three 60 Å long α- helices that contain: all sequences of HEF2 sequence, and certain HEF1 residues which are N-terminal residues (1–40), and C-terminal residues (367–432).
The crystalline structure shows that the way that HEF binds to 9-O-Ac- Neu5Ac is the same as the way HA binds to Neu5Ac. The binding parts include an α-helix, a loop and an extended strand. There are hydrogen bonds between the amino acids (Tyr127, Thr170, Gly172, Tyr227 and Arg292) and the hydroxyl-groups of the ligand, and other residues form the structural support of the receptor binding site. A unique hydrophobic pocket is present in the HEF binding site that in turn accommodates the acetyl methyl group.
Activity
Receptor binding activity Glycolipids and glycoproteins contain N-acetyl-9-O-acetylneuraminic acid (9-O-Ac- Neu5Ac) that serve as viral receptor in which HEF binds to. HEF can bind to its receptor whether or not 9-O-Ac-Neu5Ac is attached by an α-2,3 or α-2,6 linkage to the next galactosyl residue. However, host specificity can be affected by terminal N-acetylneuraminic acid (Neu5Ac) and the glycosidic linkage of Neu5Ac. Influenza C virus can recognize 9-O-Ac-Neu5Ac on the surface of different cells due to its unique receptor specificity.
Receptor hydrolysis (esterase) activity The receptor hydrolase activity of HEF aids in the release of virus particles from an infected cell using esterase enzyme that cleaves acetyl from the C9 position of terminal 9-O-Ac-Neu5Ac. The esterase activity of HEF which is part of serine hydrolase class includes a nucleophilic attack of the hydroxyl group (OH) of a serine amino acid, with the help of two other amino acids (histidine and aspartic acid), on the carbonyl group of the substrate. Basic histidine enhances the reactivity of serine by polarizing and deprotonating its hydroxyl group. Along with that, aspartic acid polarizes histidine.
X-ray crystallography of the crystalline structure of HEF showed that serine 57, aspartic acid 352 and histidine 355 are the important amino acids for the esterase activity. Also, early studies showed that mutation in Ser57 and His355 residues can completely stop the esterase activity of HEF.
Membrane fusion activity The membrane fusion activity between, the viral envelope and endocytic vesicles of host cell, is important to help the virus inject their genome into the cytoplasm of the cell. In order to activate membrane fusion, Cleaving the precursor proteins HEF0 and HA0 into the subunits into the subunits HEF1 and HEF2, then exposing these proteins to acidic pH must be done prior.
Acidic pH causes protonation of specific amino acids that initiate certain rearrangement of the proteins. The protonated amino acid is found to be histidine while its pKa matches the pH of endosome. Studies showed that there is about 0.7 difference in the pH value that trigger the membrane fusion activity from strain to strain of both influenza A and C.
Conformational change in HEF structure that occur at low pH results in the separation of fusion peptide from its location at the lower part of the stalk and exposing the outer surface of the molecule, so it can be inserted into the endosomal membrane. Another conformational change occur which cause the bending of the ectodomain to push the fusion peptide toward the transmembrane region. As a result of that, the virus and endosomal membranes get closer, exchanging lipids with hemifusion. Then, opening of a fusion pore and eventually complete merger of both lipid bilayers.
Folding and intracellular transport The folding of the hemagglutinin esterase protein and the way that the domains of the protein assembles contribute the transport of membrane and secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Researchers found that trimerization occurs at a point before exiting the ER. The monomers of the HE protein are folded before assembling is possible. Before the hemagglutinin esterase can report to the Golgi, it must be extensively folded and assembled.
The structure of hemagglutinin-esterase contributes to the intracellular transport. The hemagglutinin-esterase (HE) glycoprotein of influenza C virus is composed of three domains: a stem domain active in membrane fusion (F), an acetylesterase domain (E), and a receptor-binding domain (R). The protein contains eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603) in the F domain, three (positions 61, 131, and 144) in the E domain, and one (position 189) in the R domain. Oligosaccharide chains in the domains influence intracellular transport. A study showed that it was evident that glycosylation at the two sites in the F domain (positions 26 and 603), in addition to that in the E domain (position 144), is required for the HE molecule to be transported from the endoplasmic reticulum and that mutant HEs lacking one of these three sites failed to undergo the trimer assembly. Oligosaccharides are needed to maintain esterase activity in the F and R domains. If any of the domains lack an oligosaccharide chain, cell surface expression will be affected. It was found that HE monomer have acetylesterase activity because they possessed full-enzyme activity despite lack of an oligosaccharide chain. Oligosaccharide chains are important for intracellular transport, but not for fusion activity. Thus, oligosaccharide chains do not really promote membrane fusion.
S-acylation and RAFT-localization Acylation of the hemagglutinin-esterase enzyme is necessary for virus replication of influenza C virus. It was found that recombinant virus lacking the acylation site of HEF could be rescued, but viral titers were reduced by one log relative to wild type Flu C. The resulting virus particles have a regular protein composition and no changes in their morphology were obvious by electron microscopy, but their hemolytic activity is reduced indicating a defect in membrane fusion. This is in comparison to several HA protein subtypes that showed similar results.
The hemagglutinin-esterase-fusion protein has co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. The HEF protein of influenza C virus has only one stearate attached to a transmembrane cysteine. Whereas HA of influenza A and B virus are associated with membrane rafts, cholesterol- and sphingolipid-enriched nanodomains of the plasma membrane, HEF is thought to localize to the bulk phase of the plasma membrane.
Proteolytic cleavage The binding and cleavage properties of the influenza C virions hemagglutinin-esterase (CHE) protein for 9-O-acetyl groups on sialic acids have been used in various assays using whole virions.