Dengue virus (DENV) is one of the most important arthropod-borne viruses that infect humans. Approximately 400 million people are infected annually in tropical and sub-tropical regions worldwide [1]. In 2015, Sanofi Pasteur released the first commercialized and approved tetravalent vaccine that is protective against the four DENV serotypes [2].

In the context of developing a better understanding of the interaction of dengue antigen with the immune system, it is important to fully understand the structure and accessibility of specific epitopes. Particularly in the case of the dengue virus, viral capsids bud into the host endoplasmic reticulum and acquire structural proteins prM and E embedded in the membrane. The prM and E proteins are assembled as trimers as part of an immature icosahedral shell and undergo significant structural rearrangement during viral exit via the Golgi apparatus. In the Golgi, Host furin-type proteases concomitantly cleave the pr peptide triggering rearrangement of the trimers into dimers, which is necessary for infectivity of the viral particle [3]. However, maturation of DENV virions is not efficient and partly immature particles are released during DENV infection as well. It has been hypothesized that these particles function as decoys to avoid neutralization of the infectious mature particles by the immune system [4].

Recombinant antigens in the form of virus-like particles are attractive as reagents for understanding antigen-antibody interactions. Here, we investigated the molecular architecture of virus like particles (VLPs) made of dengue structural subunits, which are produced by The Native Antigen Company (Oxfordshire, UK). We found by SDS-PAGE and confirmed by Western blot that DENV-2 and DENV-3 VLPs contain a significant fraction of uncleaved prM subunits. To assess the molecular architecture and conformation of subunits in the VLP particles, we determined 3D structure of individual particles by cryo-electron tomography. The particles have a spherical (DENV-2 VLPs) or elongated (DENV-3 VLPs) outer shell with short spikes and an internal cavity (Figure 1). The spikes exhibit the same morphology as the prM/E subunits in the immature dengue viral particles. We additionally imaged and reconstructed the 3D structure of VLP particles mixed with monoclonal antibodies specific to the mature conformation of DENV. We identified bound antibodies to individual particles in subtomograms after subtraction of the VLP shell (Figure 2). Binding of the antibodies to the VLP particles was subsequently confirmed by Western blot and immunogenicity assays suggesting that these VLPs could adopt a mixed conformation with mature dimers and immature trimers on the same particles, as previously described for DEN virions or adopt a specific new conformation of mature prME specific to VLPs.

This study was funded by Sanofi Pasteur using equipment installed at Sanofi Pasteur.

References

[1] Bhatt S., Gething P. W., Brady O. J., et al. (2013) Nature, 496, 504–507.

[2] Hadinegoro, S.R. et al. (2015) N Engl J Med., 373, 1195–206.

[3] Kuhn R. J., Zhang W., Rossmann M. G., et al. (2002) Cell, 108, 717–725.

[4] Rodenhuis-Zybert IA1, Wilschut J, Smit JM. (2011) Trends Microbiol., 19, 248-254.

Figures:

Figure 1: Cryo-electron micrograph of DENV-2 VLPs particles collected at ~20 e-/A2 and 3-μm defocus. The 2-D projections show a spherical morphology of VLPs with short spikes.

Figure 2: Cryo-electron micrograph of DENV-3 VLPs particles collected at ~20 e-/A2 and 3-μm defocus. The 2-D projections show an elongated morphology of VLPs with short spikes.

Figure 3: A typical Z-slice through a reconstructed and denoised tomogram of DENV-3 VLP particles. Cuts through the particles indicate their elongated conformation depending on their orientation (circular from the top view and rectangular from the side view). The arrows indicate bound antibodies to the shell of VLPs.

Figure 4: 3-D rendering of an individual DENV-3 particle extracted from a tomogram (grey). Densities of bound and unbound antibodies are depicted in yellow.

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/visualization-of-dengue-virus-like-particles-interacting-with-antibodies/