A Dynamic Landscape for Antibody Binding Modulates Antibody-Mediated Neutralization of West Nile Virus

Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this “multiple-hit” perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus “breathing” in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization

Proteotype profiling unmasks a viral signalling network essential for poxvirus assembly and transcriptional competence

To orchestrate context-dependent signalling programmes, poxviruses encode two dual-specificity enzymes, the F10 kinase and the H1 phosphatase. These signalling mediators are essential for poxvirus production, yet their substrate profiles and systems-level functions remain enigmatic. Using a phosphoproteomic screen of cells infected with wild-type, F10 and H1 mutant vaccinia viruses, we systematically defined the viral signalling network controlled by these enzymes. Quantitative cross-comparison revealed 33 F10 and/or H1 phosphosites within 17 viral proteins. Using this proteotype dataset to inform genotype-phenotype relationships, we found that H1-deficient virions harbour a hidden hypercleavage phenotype driven by reversible phosphorylation of the virus protease I7 (S134). Quantitative phosphoproteomic profiling further revealed that the phosphorylation-dependent activity of the viral early transcription factor, A7 (Y367), underlies the transcription-deficient phenotype of H1 mutant virions. Together, these results highlight the utility of combining quantitative proteotype screens with mutant viruses to uncover proteotype-phenotype-genotype relationships that are masked by classical genetic studies

Temperature-dependent folding allows stable dimerization of secretory and virus-associated E proteins of Dengue and Zika viruses in mammalian cells

Dengue and Zika are two of the most important human viral pathogens worldwide. In both cases, the envelope glycoprotein E is the main target of the antibody response. Recently, new complex quaternary epitopes were identified which are the consequence of the arrangement of the antiparallel E dimers on the viral surface. Such epitopes can be exploited to develop more efficient cross-neutralizing vaccines. Here we describe a successful covalent stabilization of E dimers from Dengue and Zika viruses in mammalian cells. Folding and dimerization of secretory E was found to be strongly dependent on temperature but independent of PrM co-expression. In addition, we found that, due to the close relationship between flaviviruses, Dengue and Zika viruses E proteins can form heterodimers and assemble into mosaic viral particles. Finally, we present new virus-free analytical platforms to study and screen antibody responses against Dengue and Zika, which allow for differentiation of epitopes restricted to specific domains, dimers and higher order arrangements of E