Long Range Communication in the Envelope Protein Domain III and Its Effect on the Resistance of West Nile Virus to Antibody-mediated Neutralization

Abstract

The envelope protein domain III (ED3) of West Nile virus is the major virus-specific neutralization domain and harbors most of the critical mutations that induce resistance against antibody-mediated neutralization. We investigated the molecular mechanisms of neutralization resistance by studying the biophysical perturbations of monoclonal antibody (mAb)-resistant mutations on ED3 wild type. Our results showed that although the solution structure between ED3 wild type and mutants was preserved, the mutations that confer the highest degree of resistance to mAbs showed low protein stability and high local dynamic motions. Interestingly, the latter was observed in regions outside the mutation sites, indicating long range communications within ED3. Thus, we hypothesized that the mechanisms involved in resistance to mAb neutralization may include, in addition to mutations in the epitope, long range effects among distant structural elements. This hypothesis is consistent with reported mutations in other flaviviruses whose surfaces are not exposed for the interaction with other macromolecules, yet they confer mAb neutralization resistance.The genus Flavivirus of the family Flaviviridae consists of a number of pathogens of major public health importance that cause diseases, including yellow fever, Japanese encephalitis, tick-borne encephalitis, dengue, and, most recently, in the Americas, West Nile virus (WNV) 2 (1). Since it was first isolated in 1999, the number of human cases reporting West Nile viral encephalitis has increased dramatically. Currently there is no human vaccine to prevent WNV infections. All studies to date indicate that neutralizing antibodies are the major mechanism of protective immunity against flaviviruses (2), as demonstrated by the effectiveness of vaccines to prevent other flavivirus diseases (yellow fever, Japanese encephalitis, and tickborne encephalitis). The primary target of virus-neutralizing antibodies is the envelope (E) protein (3). The E protein is the major structural protein on the surface of flaviviruses and consists of three distinct structural domains (ED1-3). ED1 and ED2 are the central and dimerization domains, respectively, and ED3 (amino acids 295-395 in the E protein) is the putative receptor-binding domain (4, 5). Consistent with the functional role of ED3 is its orientation in the context of the intact virion. The ED3 of WNV clearly projects above the rest of the E protein facilitating the potential interaction with other macromolecules, such as antibodies or cell receptors (6).Although all three domains in the E protein contain immunogenic sites, suggesting that there is not a single defined antigenic site, most of the experimental evidence strongly indicates that ED3 is the major immunogenic domain for virus typespecific neutralizing monoclonal antibodies (mAbs) (3). Many studies have shown that mAb binding to ED3 is most efficient at blocking virus attachment to cells (7-10), and recently it has been suggested that some anti-ED3 mAbs may also impair virus membrane fusio...

Thermodynamic Mechanism for the Evasion of Antibody Neutralization in Flaviviruses

“…Data were obtained with permission from Maillard et al. 21 The values of ΔΔ G W397 mutation were calculated using eq 4 in Methods. The structure on the right side displays the position of the mutation sites and W397.…”

“…21 Binding data for other single-site mutant ED3s from WNV or DENV-2 used in this study were obtained from the literature (Table 1). …”

“…aBinding data for the ED3 from DENV-2 against two type-specific MAbs, 5H5 and 9F16, was obtained from Hiramatsu et al, 36 Gromowski and Barrett, 24 and Pitcher et al 51 bFor the WNV ED3s K307R, K307E, Y329K, Y329F, wild-type, A365S, L312A, A369S, K310T, T330I, T332A, and T332K, binding data against type-specific MAbs 5H10, 5C5, and 3A3 was obtained with permission from Beasley and Barrett, 1 Volk et al, 13 Li et al, 19 Maillard et al, 21 and Zhang et al 9 For the WNV-ED3s E390D, H396Y, L312V, V371I, V338I, and L375I, we measured mAb binding against the same three type-specific MAbs. For each mAb, binding was measured in triplicate.…”

ANALYSIS OF DOMAIN SPECIFICITY OF THE PROTECTIVE CHIMERIC ANTIBODY ch14D5a AGAINST GLYCOPROTEIN E OF TICK-BORNE ENCEPHALITIS VIRUS

“…Это достаточно стабильный домен, до-полнительно стабилизированный одной консервативной дисульфидной связью. Рекомбинантные белки, представ-ляющие собой домены D3 некоторых флавивирусов, таких как вирус желтой лихорадки, вирус Денге, вирус западного Нила и вирус Лангат, были получены в бактериальных системах экспрессии как в виде телец включения с по-следующим рефолдингом белка (Volk et al, 2009;Elahi et al, 2014;Kulkarni et al, 2016), так и в растворимом виде в формате белков слияния (White et al, 2003;Volk et al, 2006;Maillard et al, 2008). В случае периплазматической экспрессии домены D3 вируса японского энцефалита и вируса лихорадки Денге были получены в нативной кон-формации без рефолдинга и отщепления белка-носителя (Wu et al, 2003;Lisova et al, 2007;Yang et al, 2012).…”

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

“…Although all three domains of the E protein appear to contribute to interactions with target cell ligands in different experimental systems, data that support a significant role for EIII in receptor binding come from investigations into competition between recombinant EIII molecules and infectious virions for adsorption to cell surfaces (9)(10)(11), immunological studies using neutralizing monoclonal antibodies (MAbs) targeting EIII (12), studies in which mutant viruses containing particular amino acid substitutions in EIII were generated (13)(14)(15)(16)(17)(18)(19), and investigations with dengue virus (DENV) E fusion pro-teins (6). Studies of EIII protein subunits derived from different mosquito-and tick-borne flaviviruses have identified virus-specific differences in antigenicity, surface biochemistry, and structure, which further support a role for EIII as a key determinant of ligand binding and cell tropism for individual flavivirus types (20)(21)(22)(23)(24)(25)(26)(27)(28).…”