Research paperDirect visualization of the oligomeric state of hemagglutinins of influenza virus by high-resolution atomic force microscopy
Introduction
Influenza virus is one of the most widespread pathogens worldwide, causing epidemics every year and occasionally pandemics [1]. Hemagglutinin is a viral transmembrane protein that mediates recognition of the host cell by the influenza virus and transfer of the viral genome inside the cell [2]. As hemagglutinin is exposed on the surface of the virus, it has become a target of antiviral drugs, and is also widely used for virus typing [1,3]. Thus, the structure and function of hemagglutinin is of great importance in the study of the influenza virus.
While it has a fairly uniform spatial structure, hemagglutinin is a highly variable protein in terms of primary structure. Currently, 18 subtypes of hemagglutinins have been identified for influenza virus type A; each subtype comprises dozens of related proteins. In addition to its sequence diversity, hemagglutinin also exhibits a variety of post-translational modifications that affect its functioning [1,4].
Functional activity of hemagglutinin can be represented as: i) recognition of the host cell through the binding of specific sialic acids; and ii) pH-dependent conformational change (Fig. 1A) that is required for merging the viral membrane and the membrane of the exosome of the host, leading to viral genome transfer into the cell cytoplasm [2,5,6].
The trimeric form of hemagglutinin (Fig. 1B) is considered functionally active and the predominant form within the influenza virus [4]. Recombinant hemagglutinins have been shown to exist as monomeric, trimeric, and higher oligomeric forms without defined stoichiometry [7,8]. The oligomeric state of the protein is a result of a balance of several factors: primary structure of the protein, post-translational modifications, and the method of purification. The ability to form oligomers is associated with the functional activity of hemagglutinin [8], therefore it could be a critical parameter to monitor when studying the protein.
Atomic force microscopy (AFM) is a powerful technique for the direct visualization of the oligomeric state of proteins. A unique advantage of AFM is that only a very small amount of protein, as low as 1 ng, is required. This value is not comparable to that for other techniques, such as size-exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation. AFM can visualize single molecules, and therefore can directly reveal the landscapes of sizes and shapes. Validation of the data can be achieved through statistical analysis of thousands of particles. The main complication of AFM when used to study proteins is the high resolution required. Broadening of an object is an inherent feature of AFM [[10], [11], [12], [13]]. A simple explanation is given in Fig. S1 (see Supplementary materials). When the tip of a cantilever is sliding over an object, it reaches the substrate with a delay due to physical hindrances. As proteins are usually small objects with a typical diameter of 2–5 nm, the cantilever tips have to be extremely thin, approximately 1 nm in diameter.
Several studies using high-resolution AFM have been published recently [[10], [11], [12], [13]]. This technique allows the visualization of denaturated protein chains [11], domain structures of proteins [13], and even subtle protein–protein interactions [12]. A good correlation between geometric parameters in X-ray resolved structures and AFM images has been demonstrated [[11], [12], [13]]. Here, we provide the first application of high-resolution AFM in the study of the oligomeric state of a protein. Recombinant hemagglutinins from different strains of influenza viruses were studied at neutral and slightly acidic pH, revealing a pH-dependent heterogeneity of particle size. Further, we have studied the effect of the addition of hemagglutinin ligands, a monoclonal antibody and a DNA aptamer, on the oligomeric state of the protein, providing convincing data of ligand-induced alteration of the oligomeric state of hemagglutinin.
Section snippets
Materials and methods
Recombinant hemagglutinins were purchased from Abcam (H1N1 strain, ab69741; H3N2 strain, ab69749; H5N1 strain, ab190125; H7N9 strain, ab190421; and H9N2 strain, ab67740; USA). The hemagglutinins were expressed using baculovirus infected insect cells that provides all necessary post-translational modifications, such as extensive glycosylation and cleavage of the peptide bond during normal protein processing. Monoclonal antibody against hemagglutinin H3 was purchased from Roche (anti-HA [12CA5]
Visualization of hemagglutinins from different influenza strains
Recombinant hemagglutinins from influenza strains H1N1, H3N2, H5N1, H7N9, and H9N2 were studied with AFM. The distribution histograms of height and volume extracted from the images for the samples at pH 7.6 are shown in Fig. 2. Generally, objects are globular without noticeable asymmetry. Special comments are required to explain the detected size of the particles.
Hemagglutinin monomer (supposing the same conformation as within the trimer; Fig. 1) can be approximated as a cylinder with a 12 nm
Conclusions
High-resolution AFM was revealed to be a robust technique for the characterization of oligomeric proteins. Distribution histograms readily showed changes in the oligomeric state of influenza hemagglutinins. Hemagglutinin oligomers are unstable structures; the dynamic balance is shifted with changes in pH or the addition of ligands. Antibody binding shifted the balance to small associates, while the addition of a DNA aptamer induced the formation of large associates.
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. N. Barinov performed AFM experiments. Nikita Ivanov prepared samples for the experiments. A. Kopylov designed the study. D. Klinov handled and interpreted the AFM data. E. Zavyalova generalized the results and wrote the article.
Funding sources
This work was supported by the Russian Science Foundation [grant no. 15-13-00033].
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