In:
PLOS ONE, Public Library of Science (PLoS), Vol. 17, No. 3 ( 2022-3-1), p. e0264643-
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.
Type of Medium:
Online Resource
ISSN:
1932-6203
DOI:
10.1371/journal.pone.0264643
DOI:
10.1371/journal.pone.0264643.g001
DOI:
10.1371/journal.pone.0264643.g002
DOI:
10.1371/journal.pone.0264643.g003
DOI:
10.1371/journal.pone.0264643.g004
DOI:
10.1371/journal.pone.0264643.g005
DOI:
10.1371/journal.pone.0264643.s001
DOI:
10.1371/journal.pone.0264643.s002
DOI:
10.1371/journal.pone.0264643.s003
DOI:
10.1371/journal.pone.0264643.s004
DOI:
10.1371/journal.pone.0264643.s005
DOI:
10.1371/journal.pone.0264643.s006
DOI:
10.1371/journal.pone.0264643.s007
DOI:
10.1371/journal.pone.0264643.s008
DOI:
10.1371/journal.pone.0264643.s009
Language:
English
Publisher:
Public Library of Science (PLoS)
Publication Date:
2022
detail.hit.zdb_id:
2267670-3
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