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

Abstract

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.

Multiple Virtual Screening Strategies for the Discovery of Novel Compounds Active Against Dengue Virus: A Hit Identification Study

“…The tip of DII (Y' site), a conserved region among flavivirus genus such as West Nile virus, Japan encephalitis virus, Yellow fever virus, or Zika virus that immerses into the lipid bilayer of host endosomal membrane, has been suggested as the binding site for flavonoids, possibly interfering with the viral attachment and fusion [42,57,58]. In addition, the region between DI and DIII (X' and Y' sites), a promising target for vaccine development [59,60], was also used for drug discovery [50,54]. From an in vitro and in silico study [54], a flavanone derivative (5-hydroxy-7-methoxy-6-methylflavanone, FN5Y) has been shown to interrupt the progress of the disease at the early stage of infection, targeted at E protein at the four sites ( Figure 1).…”

Oligomeric state of the ZIKV E protein defines protective immune responses

“…Here, to investigate if ZIKV rE dimers are better subunit vaccine antigens than monomers, we expressed ZIKV rE variants (aa1-402) that are monomers (rE M ) or stable dimers (rE D ) under physiological conditions. We generated stable ZIKV rE D proteins as previously described for DENV E protein by introducing a disulfide bridge (A264C) in the E-domain II interactive region of the homodimer 9,10 ( Fig.1A ). The oligomeric state of the purified rE M and rE D proteins were confirmed by protein gel electrophoresis ( Fig.1B ).…”

Zika virus-like particles bearing covalent dimer of envelope protein protect mice from lethal challenge

“…E-homodimer stability was proved several times to be affected by temperature: dimerization is favoured at 28˚C and reduced at 37°C, hence physiological temperature is another element that contributes to impair dimers exposure to the immune system (39). But 28˚C is also a crucial temperature for expression and secretion of E protein, both in the soluble form but also as part of VLPs (28). Since this temperature is not compatible with genetic vaccination approaches, in which DNA or RNA encoding the antigen is administered and the antigen is then produced by the host cells at physiological temperature, the full potential of our antigens as candidate vaccines was evaluated in a protein-based vaccination approach.…”

“…In order to further characterise the types of antibodies elicited by our cvD antigens, we used a recently developed cytofluorimetry assay of cells displaying dimers of ZIKV sE protein (paper in submission). In this assay, the C-terminus of sE is fused to the transmembrane and cytosolic tail of the type-I trans-membrane protein MHC-Iα for plasma membrane display of the protein, as previously reported (28). This assay has the potential to discriminate antibodies binding exclusively to dimeric E on the basis of the pH-dependent mobility of E protein: at pH7, that resembles the neutral extracellular environment, the protein is in a dimeric conformation but at pH6, mimicking the conformational changes that occur in the acidic endosome vesicles during infection, it moves to a pre-fusion monomeric conformation.…”