Hemagglutinin esterase

HEs helps in the attachment and destruction of certain sialic acid receptors that are found on the host cell surface.

[2] 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.

HEF proteins have been tested to be high-temperature and low-pH resistant and are the primary source of virulence in viruses.

Acylation of the hemagglutinin-esterase has shown to play an essential role in virus particle assembly replication.

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.

[4] "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".

[2] 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.

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.

Influenza C virus can recognize 9-O-Ac-Neu5Ac on the surface of different cells due to its unique receptor specificity.

[2] 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.

Also, early studies showed that mutation in Ser57 and His355 residues can completely stop the esterase activity of HEF.

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.[2] 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.

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.[2] 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.

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.

[2] Polybasic cleavage sites that are present in HA of highly pathogenic avian influenza A viruses and processed by the ubiquitous protease furin are not found in any HEF protein.

[8] Like other hemagglutinating glycoproteins of influenza viruses HEF is S‐acylated, but only with stearic acid at a single cysteine located at the cytosol‐facing end of the transmembrane region.

HEF trimers on the surfaces of both spherical and filamentous particles are arranged in a reticular structure that has been described to consist mainly of hexagons.

Glycosylation is crucial for proper folding because it protects it from proteolytic degradation from the host cell and is important for the presentation of antigenic epitopes.

There is a site at position 589 on the crystallized structure that is not glycosylated and this may be due close location to the membrane-spanning regions and cannot be accessed by oligosaccharide transferase.

[2] In HEF1, 12/15 cysteine residues form 6 intrachain disulfide linkages that stabilizes the globular head domain.

The rest of the cysteine residues form interchain disulfide bonds with HEF in the ectodomain area, near the bottom of the trimer.

These disulfide bonds in HEF2 allows the subunit to perform large conformational changes that catalyze membrane fusion.

Two of the cysteine residues are not required for proper folding and function of HEF and/or they do not form a disulfide linkage in the mature protein located at the connection hinge.

[2] During translocation of HEF into the lumen of the ER, the N-terminal signal peptide is cleaved, and carbohydrates are attached.

Later on, a fatty acid chain is attached to the cysteine located on the end of the transmembrane region and HEF is cleaved into 2 subunits, this process is essential for virus replication.

In influenza A, the rearrangement of hydrophobic sequences at the N-terminus of subunit HEF2 becomes exposed and induces the fusion of the viral envelope with the membrane of the target cell.