Abstract
Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4’ is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation.
Highlights
Dengue virus (DENV) structure consists of a spherical particle of 50 nm diameter formed by a lipid bilayer associated with two structural proteins, the membrane (M) and envelope (E) proteins [3, 4], surrounding viral NC, which is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein [4]
To determine the optimal conditions for in vitro assembly of DENVC nucleocapsid-like particles (NCLPs) in solution as well as the effect of the oligonucleotide size in the particles formed, the recombinant DENVC was incubated with single-stranded DNA oligonucleotide (ssDNA) of different lengths, and the formation of particles was evaluated by small angle X-ray scattering (SAXS), Rayleigh light scattering, and fluorescence anisotropy (Fig 1)
The results showed that while for samples prepared in the absence of oligonucleotide only amorphous protein aggregates were observed, particles similar for those observed in atomic force microscopy (AFM) images obtained on negatively charges mica were visualized for the samples in the presence of 25-mer ssDNA applied on neutralized surface (S2 Fig)
Summary
Several pieces of evidence have suggested that viruses and viral capsids are highly dynamic structures [1] Analytical techniques such as atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray diffraction, and cryo-electron microscopy (Cryo-EM), have made important contributions to the understanding of the structure and assembly of viral nucleocapsid (NC) [1, 2], an important and complex event in the viral cycle. Despite Cryo-EM studies revealed structural details of mature flavivirus particles, including the organization of E and M proteins on the virus surface, structural information on the NC is still lacking [4–10] This led flaviviruses’ NC to be considered as an amorphous structure lacking symmetry, unlike alphaviruses’ NC, which have a highly ordered geometry with 240 copies of the capsid protein assembled with a T4 icosahedral symmetry [11–13]. Viral genome recognition by C protein as well as its interaction with cellular and viral components seem to be very well coordinated, making it difficult to isolate NC from infected cells [19–21]
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