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The Power and Limitations of Influenza Virus Hemagglutinin Assays

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The Power and Limitations of Influenza Virus Hemagglutinin Assays ISSN 00062979, Biochemistry (Moscow), 2017, Vol. 82, No. 11, pp. 12341248. © Pleiades Publishing, Ltd., 2017. Original Russian Text © N. B. Ustinov, E. G. Zavyalova, I. G. Smirnova, A. M. Kopylov, 2017, published in Biokhimiya, 2017, Vol. 82, No. 11, pp. 15771592. REVIEW The Power and Limitations of Influenza Virus Hemagglutinin Assays N. B. Ustinov, E. G. Zavyalova*, I. G. Smirnova, and A. M. Kopylov Lomonosov Moscow State University, Faculty of Chemistry, 119991 Moscow, Russia; Email: [email protected] Received May 12, 2017 Revision received August 8, 2017 Abstract—Influenza virus hemagglutinins (HAs) are surface proteins that bind to sialic acid residues at the host cell surface and ensure further virus internalization. Development of methods for the inhibition of these processes drives progress in the design of new antiviral drugs. The state of the isolated HA (i.e. combining tertiary structure and extent of oligomerization) is defined by multiple factors, like the HA source and purification method, posttranslational modifications, pH, etc. The HA state affects HA functional activity and significantly impacts the results of numerous HA assays. In this review, we ana lyze the power and limitations of currently used HA assays regarding the state of HA. DOI: 10.1134/S0006297917110025 Keywords: influenza virus, surface antigens, influenza hemagglutinin, inhibitors of hemagglutination, monoclonal antibod ies, ELISA Influenza is one of the most common infectious dis endosome with subsequent nucleoprotein release from eases: 510% of adults and 2030% children are infected the endosome (HA and M2 protein), and detachment of with influenza each year. Influenza infection itself lasts the newly synthesized virus particles from the host cell only for several days, but the high risk for human health surface (neuraminidase, NA) [35]. Surface proteins are comes from secondary infections that develop in influen the major targets for existing antiviral drugs: Arbidol zadamaged tissues and from complications that result (umifenovir) inhibits HA at the step of the virus mem from preexisting conditions (pregnancy, immunodefi brane fusion with the endosome [68]; amantadine and ciency) or disorders (diabetes, obesity, cardiovascular dis rimantadine inhibit the M2 protein [9, 10]; zanamivir, orders). The annual number of influenzaassociated oseltamivir, and peramivir inhibit NA [912]. Rapid deaths is 250,000 to 500,000 [1, 2]. microevolution of the influenza virus results in an Influenza virus belongs to the Orthomyxoviridae extremely high variability of its surface proteins, which viral family, the most dangerous member of which is ensures rapid selection of strains resistant to the action of influenza A virus. Influenza virus consists of a mem existing antiviral drugs. Presentday influenza virus braneenveloped ribonucleoprotein core. Its genome is strains are already resistant to amantadine and rimanta segmented and contains eight antisense RNAs coding for dine [13]. For this reason, active search for new antiviral 11 viral proteins. Viral surface proteins provide virion agents, including HA inhibitors, has to be continued [14, interaction with the host cell plasma membrane (hemag 15]. HA is the most promising target for the new drugs, glutinin, HA), fusion of the virus membrane with the because it is responsible for the very first stage of viral infection, namely, virus attachment to the host cell. Since HA is not an enzyme, potential HA inhibitors should Abbreviations: a.a., amino acid residue; ELISA, enzymelinked either inhibit low pHinduced conformational transitions immunosorbent assay; ER, endoplasmic reticulum; GA, Golgi of HA or prevent formation of the complex between HA apparatus; HA, hemagglutinin; HA0, hemagglutinin precursor; and cell surface molecules (including oligosaccharides). HA1, hemagglutinin subunit 1; HA2, hemagglutinin subunit 2; HAI, hemagglutination inhibition assay; IEP, immunoelec The main obstacle in the design of new HA inhibitors is trophoresis; MAb, monoclonal antibody; NA, neuraminidase; that they cannot be characterized by standard enzymo RIA, radioimmunoassay; RIP, radioimmunoprecipitation; SA, logical methods and should be tested in a carefully chosen sialic acid; SRID, single radial immunodiffusion. in vitro test system. In our review, we discuss the capabil * To whom correspondence should be addressed. ities and limitations of the currently used test systems. 1234 FUNCTIONAL ACTIVITY OF HEMAGGLUTININS 1235 It is not surprising that influenza virus HA has been 23] (depending on the virus subtype); (ii) glycosylation actively studied – dozens of reviews have been published [24] and removal of the Nterminal signal peptide [25] in that search for the correlation between the HA structure the ER; (iii) palmitoylation of the terminal COOHgroup and influenza virus virulence. Because HA is a highly vari in the early GA cisterns [26]; and (iv) cleavage in the able protein, data on its structure and functions have been transGolgi network with the formation of two polypep accumulated in large dynamic databases, such as the tide chains connected by a disulfide bond [27, 28]. HAs of Influenza Research Database (www.fludb.org) and GisAid the highly pathogenic avian influenza virus H5 and H7 (www.platform.gisaid.org). Several recent reviews summa subtypes are cleaved by cellular subtilisinlike proteases rize structural properties of HAs from different viral strains [29]. In other virus subtypes HAs are cleaved predomi [16], effects of posttranslational modifications on the virus nantly by the transmembrane type II serine proteases [30]. virulence [17], HA functional properties (especially, its The HA molecule in the virus membrane consists of pHdependent conformational transitions) [16, 18], and the hydrophilic ectodomain exposed at the membrane data on the mapping of viral antigenic determinants [19]. outer side, one small transmembrane domain (2428 a.a.), The properties of HA outside the virus are affected and one cytoplasmic domain (1055 a.a.) [31]. Crystal by multiple factors. The first is the HA primary structure structures for the ectodomains of mature HA [32, 33] and diverse posttranslational modifications. Multiple (Fig. 1b) and its precursor (HA0) (Fig. 1a) [34] have been virus mutants have been characterized, and the obtained solved. The ectodomain consists of distal globular and information was used to identify phylogenetic relation proximal fibrillar fragments corresponding to HA subunits ships between these viruses (see GisAid database and 1 and 2 (HA1 and HA2), respectively. The surface of the [20]). The second and the third factors are HA conforma globular fragment bears HA antigenic determinants [35] tion and extent of oligomerization. If the primary struc and serves as a binding site for sialic acids (SAs) of the host ture and posttranslational modifications are determined cell surface [36]. The fibrillar fragment is formed by six α by the virus strain and the host cell, HA conformation helices (two helices from each HA2 molecule); three of and extent of oligomerization (defined by us as the “HA them are wound in a lefthanded superhelix. state”) depend on the method for HA isolation and some In addition to the supercoiling of the ectodomain fib external parameters, such as pH, buffer composition, etc. rillar fragment, HA quaternary structure (Fig. 2) is stabi HA forms functionally active trimers in the virus lized by noncovalent interactions between monomer sub envelope [19]. Isolated HA can exist either as an active units. It was found that stability of the HA trimer in the trimer or inactive oligomers composed of a varying num pandemic A(H1N1)pdm09 strain is determined by amino ber of HA monomers. The number of HA molecules in acids at positions 205 and 402 in the HA1 and HA2 sub the oligomer depends greatly on posttranslational modifi units, respectively (Fig. 2) [37]. Another driving force for cations, especially, polysaccharide attached to Asn trimerization is the orientation effect of the membrane residues in HA [17]. anchored Cterminal sequence of HA2. Thus, both full This existence of diverse HA states often causes ambi size HA [38] and HA with the Cterminal glycosylphos guities in the estimates of the activity of HA inhibitors or phatidylinositol anchor in place of the transmembrane and affinity of other HAinteracting molecules, e.g. antibodies cytoplasmic domains [39] form trimers, whereas isolated (see section “Variety of antiHA antibodies” of this ectodomain is incapable of spontaneous trimerization [38]. review). Thus, IC50 values for the same inhibitor might dif Disulfide bonds between the subunits are another impor fer ten times depending on the HA state. tant factor that, together with protein–lipid interactions, In this article, we review procedures for HA isolation affects the structural stability and functional activity of HA and methods for the estimation of its functional activity. molecules. Formation of disulfide bridges between cysteine Special attention is given to the HA states and test systems residues of the transmembrane and cytoplasmic domains is that could be used for the development of molecules that critical for protein trimerization [40]. Interestingly, isolat would recognize these states (e.g. aptamers and antibod ed HA1 molecules, but not ectodomains, can form biolog ies capable of blocking HA interaction with its ligands). ically active trimers [41]. Trimerization in the absence of transmembrane domains is initiated by the interactions between the Ile3–Cys4–Ile5
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