Recombinant Influenza C virus Hemagglutinin-esterase-fusion glycoprotein (HE), partial

What is the structural composition of the Influenza C virus Hemagglutinin-esterase-fusion glycoprotein?

The HEF glycoprotein is synthesized as a single 80K glycosylated polypeptide precursor (HEF0) that undergoes proteolytic cleavage to produce disulfide-linked subunits HEF1 and HEF2. Each HEF monomer consists of three functional domains: the receptor-binding domain (R) that recognizes receptors containing 9-O-acetyl sialic acid, an esterase domain (E) with receptor-destroying 9-O-acetylesterase activity, and a fusion domain (F) formed by segments of both HEF1 and HEF2 . The HEF protein forms trimers that arrange in distinctive hexagonal arrays on the virion surface .

How does the HEF protein differ from the hemagglutinin (HA) and neuraminidase (NA) proteins of influenza A and B viruses?

Unlike influenza A and B viruses that utilize separate proteins (HA and NA) for receptor binding/fusion and receptor destruction, influenza C virus employs a single multifunctional glycoprotein (HEF) that encompasses all three activities. The HEF protein recognizes 9-O-acetylated sialic acid receptors, which differ from the receptors used by influenza A and B viruses by the addition of an acetyl group at the 9-O position of the glycerol side chain of sialic acid . Evolutionarily, HEF proteins of influenza C are remarkably stable, with only approximately 5% of amino acid residues not conserved across all lineages, contrasting with the highly variable HAs of influenza A and B .

What expression systems are most effective for producing recombinant HEF protein for structural studies?

Multiple expression systems have been employed for HEF production, but with varying degrees of success regarding proper folding and transport. When expressed alone using recombinant simian virus 40, the 80K HEF precursor is synthesized but fails to convert to the 100K form (indicative of proper folding) and is not transported to the cell surface . The vaccinia virus expression system shows improved results, with complete conversion to the 100K form, suggesting proper disulfide bond formation, though cell surface transport remains impaired . For functional studies, co-expression with other viral components, particularly the M protein which may act as a chaperone, appears necessary for proper folding and transport of HEF .

How can researchers verify the proper folding of recombinant HEF protein?

Proper folding of the HEF protein can be assessed through:

• SDS-PAGE analysis under reducing and non-reducing conditions: Properly folded HEF exhibits different electrophoretic mobilities—approximately 100K under non-reducing conditions and 80K under reducing conditions—due to intramolecular disulfide bonds affecting protein conformation .

• Timing of conformational changes: In infected cells, the 100K form appears approximately 10 minutes after synthesis, with transport to the cell surface occurring around 60 minutes post-synthesis .

• Functional assays: Testing receptor-binding activity (hemagglutination), esterase activity (using synthetic substrates like p-nitrophenyl acetate), and fusion activity (syncytia formation or lipid mixing assays) provides comprehensive validation of proper HEF folding and function.

How does the conformation of HEF in situ differ from isolated trimeric ectodomains?

Electron cryotomography and subtomogram averaging studies reveal that the in situ conformation of HEF on the viral surface differs substantially from the structure determined by X-ray crystallography of isolated ectodomains. The membrane-distal domains of HEF exhibit significant splaying in the virus surface lattice, creating an open cavity along the trimer axis that exposes HEF2 . This conformational difference appears necessary for lateral interactions between HEF trimers that form the characteristic hexagonal lattice . The splayed conformation resembles an early dilated fusion intermediate and may facilitate low-pH transitions required for membrane fusion, suggesting functional significance beyond merely structural organization .

What molecular interactions drive the formation of hexagonal HEF arrays on the virion surface?

The hexagonal arrangement of HEF on the influenza C virus surface is mediated by:

• Lateral interactions between the ectodomains of adjacent HEF trimers, particularly involving two specific regions that were identified through mutation studies to be critical for virus rescue .

• Interactions between the short cytoplasmic tail of HEF (Arg-Thr-Lys) and the matrix protein (CM1), which stabilize the HEF clusters. Deletion of this tail reduces both virus titer and the extent of hexagonal HEF arrays .

These interactions lead to approximately 250-nm sized HEF clusters at the plasma membrane that are insensitive to cholesterol extraction and cytochalasin treatment, indicating that neither lipid rafts nor the actin cytoskeleton are essential for cluster formation .

What experimental approaches can determine the receptor-binding specificity of HEF?

Researchers can employ several complementary approaches to characterize HEF receptor specificity:

• Solid-phase binding assays using purified HEF and immobilized glycans with varying O-acetylation patterns.

• Surface plasmon resonance (SPR) or biolayer interferometry (BLI) to measure binding kinetics to various sialic acid derivatives.

• Glycan array screening to identify preferred receptors from hundreds of naturally occurring glycan structures.

• Cell-based binding assays with cells expressing different sialic acid modifications, quantified by flow cytometry.

• Hemagglutination inhibition assays using erythrocytes with naturally occurring or enzymatically modified sialic acids.

These methods together provide a comprehensive profile of the receptor specificity of recombinant HEF proteins.

How can the esterase activity of recombinant HEF be measured and characterized?

The receptor-destroying esterase activity of HEF can be quantified through:

• Colorimetric assays using p-nitrophenyl acetate (pNPA) as a synthetic substrate, which releases p-nitrophenol upon hydrolysis that can be measured spectrophotometrically.

• Fluorometric assays with 4-methylumbelliferyl acetate (4-MUA), which produces fluorescent 4-methylumbelliferone upon cleavage.

• Direct measurement of O-acetyl group removal from 9-O-acetylated sialic acids using mass spectrometry or NMR spectroscopy.

• Biological assays measuring elution of virus or HEF protein from erythrocytes or cells over time.

Kinetic parameters (Km, Vmax) for different substrates provide insights into substrate specificity and enzymatic efficiency.

What reverse genetics approaches enable the generation of recombinant influenza C viruses with modified HEF?

Reverse genetics systems for influenza C virus have been successfully established using plasmid-based methods. A typical approach includes:

• Cloning all seven full-length genomic segments of influenza C virus (including the HEF-encoding segment) into expression vectors under control of RNA polymerase I promoters.

• Co-transfection of these plasmids along with plasmids expressing viral polymerase proteins into suitable cells (e.g., 293T cells grown on poly-lysine plates).

• Culture with optimal TPCK-trypsin concentration (approximately 0.25 μg/ml for 293T cells) to enable HEF cleavage required for infectivity.

This system typically yields virus titers of 10^3 to 10^4 PFU/ml at 8-10 days post-transfection , enabling the production of viruses with targeted mutations in HEF for functional studies.

Which regions of the HEF protein are critical targets for structure-function mutagenesis studies?

Based on current research, several regions of HEF are particularly informative targets for mutagenesis:

• The receptor-binding pocket within the R domain: Mutations here affect receptor specificity and binding affinity.

• The catalytic triad of the esterase domain: Typically involves a serine-histidine-aspartic acid configuration essential for enzymatic activity.

• The fusion peptide region in HEF2: Critical for membrane fusion activity.

• The interface regions mediating lateral interactions between HEF trimers: Two specific regions have been identified that, when mutated, prevent rescue of infectious virus particles .

• The short cytoplasmic tail (Arg-Thr-Lys): Substitution of the basic amino acids with hydrophobic (Ile) or acidic residues (Glu) affects protein folding .

• The membrane-proximal regions involved in the conformational changes needed for membrane fusion.

Systematic mutagenesis of these regions provides insights into structure-function relationships crucial for HEF activity and virus replication.

What is the role of HEF in influenza C virus assembly and budding?

HEF plays multiple critical roles in virus assembly and budding:

• Formation of hexagonal lattices: The lateral interactions between HEF trimers create distinctive hexagonal arrays on the virus surface that act as a driving force for membrane curvature and budding .

• Independent particle formation: Expression of HEF alone can cause the release of membrane-enveloped particles with hexagonal HEF arrays, demonstrating its intrinsic ability to vesiculate membranes .

• Interaction with matrix proteins: The cytoplasmic tail of HEF interacts with the matrix protein (CM1), which stabilizes HEF clusters and affects particle morphology .

Electron cryotomography studies reveal that spherical influenza C particles can form with or without a dense matrix layer and that HEF organization into hexagonal lattices occurs regardless of internal structure, though the matrix layer influences particle morphology and genome packaging .

How do mutations in HEF affect particle formation and virus infectivity?

The effects of HEF mutations on particle formation and infectivity vary depending on the region altered:

• Cytoplasmic tail deletions: Removal of the short cytoplasmic tail reduces virus titer and hexagonal HEF arrays, indicating its importance in stabilizing interactions with the matrix protein .

• Surface mutations affecting the closed conformation: Specific mutations prevent virus rescue entirely, while others reduce virus titers and decrease the number of HEF clusters in virions .

• Mutations in regions mediating contacts between trimers: Alterations to two key regions that facilitate inter-trimer interactions in the hexagonal lattice completely prevent rescue of infectious virus particles .

• Mutations affecting lateral interactions: These often cause intracellular trafficking defects, highlighting the dual role of certain HEF regions in both assembly and transport .

These findings collectively indicate that HEF's ability to form organized structures on the viral surface is intrinsically linked to successful virus assembly and infectivity.

What cutting-edge imaging techniques are most informative for studying HEF distribution and organization?

Several advanced imaging approaches provide valuable insights into HEF structure and organization:

• Super-resolution microscopy: Reveals approximately 250-nm sized HEF clusters at the plasma membrane that are insensitive to cholesterol extraction and cytochalasin treatment .

• Electron cryotomography: Allows visualization of the pleomorphic virus particles, including both spherical and filamentous forms, showing the hexagonal lattice arrangement of HEF on the virus surface .

• Subtomogram averaging: Provides detailed structural information about the in situ conformation of HEF in the virus surface lattice, revealing conformational differences compared to isolated protein structures .

• Correlative light and electron microscopy (CLEM): Enables tracking of fluorescently labeled HEF during virus assembly and correlating this with ultrastructural information.

These complementary techniques provide multi-scale information from molecular to viral particle levels.

How can researchers effectively detect and quantify influenza C virus infection in experimental and clinical samples?

A comprehensive approach to influenza C virus detection and quantification includes:

• Real-time RT-PCR: Targeted assays using primers designed for conserved regions of the matrix (M) gene provide sensitive detection. Primer pairs such as INFC-M-For/INFC-M-Rev with an MGB probe (INFC-M-Probe) have been developed to amplify a 64-bp region of the matrix gene .

• Immunofluorescence assays: Using antibodies against the HEF protein to visualize infected cells.

• Virus titration: Plaque assays or endpoint dilution assays (TCID50) in suitable cell lines with trypsin supplementation.

• Sequence analysis: Full or partial sequencing of the M and HEF genes using specific primers designed for amplification and sequencing .

• Hemagglutination assays: Utilizing the receptor-binding property of HEF to agglutinate erythrocytes as a quantitative measure of virus concentration.

Real-time RT-PCR is particularly valuable for clinical and epidemiological studies, as influenza C infections are often under-diagnosed due to their typically mild nature and the lack of convenient detection methods .

What experimental approaches show promise for developing vaccines targeting influenza C virus HEF?

Several approaches for developing influenza C virus vaccines targeting HEF show research potential:

• Recombinant influenza viruses as vectors: Studies have demonstrated that recombinant influenza viruses carrying antigenic epitopes from other pathogens can induce robust immune responses. Similar approaches could be adapted for influenza C antigens .

• Subunit vaccines: Recombinant HEF protein produced in suitable expression systems could be formulated as subunit vaccines.

• Virus-like particles (VLPs): Expression of HEF alone can generate membrane-enveloped particles resembling virions , which could potentially serve as non-infectious VLP vaccines.

• Chimeric constructs: Since HEF contains receptor-binding, receptor-destroying, and fusion domains in a single protein, chimeric constructs incorporating key epitopes from each functional domain might elicit broad protective immunity.

• DNA or mRNA vaccines: Genetic vaccines encoding the HEF protein could induce both humoral and cellular immune responses.

Preclinical evaluation would need to assess humoral (antibody) responses, mucosal immunity, and cellular immune responses to determine optimal formulation and delivery routes.

What are the challenges and considerations in designing inhibitors that target the multiple functions of HEF?

Developing inhibitors against HEF presents unique challenges due to its multifunctional nature:

• Targeting receptor binding: Inhibitors must specifically block interaction with 9-O-acetylated sialic acids without affecting host sialic acid recognition systems.

• Esterase inhibition: Compounds targeting the receptor-destroying esterase activity must be specific for the 9-O-acetylesterase function of HEF without affecting host esterases.

• Fusion inhibition: Peptides or small molecules that prevent the conformational changes required for fusion activity could target the splayed conformation observed in HEF clusters .

• Disrupting lattice formation: Molecules that interfere with lateral interactions between HEF trimers could potentially inhibit the hexagonal lattice formation essential for virus budding .

• Dual-targeting approaches: Given HEF's multifunctionality, compounds targeting multiple functions simultaneously might offer advantages, although this increases design complexity.