ABSTRACT
Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV-rNP). OROV-rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV-rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV-rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV-rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses.
Subject(s)
Genome, Viral , Nucleoproteins/chemistry , Protein Multimerization , RNA, Viral/chemistry , Simbu virus/chemistry , Viral Proteins/chemistry , Humans , Nucleoproteins/genetics , Nucleoproteins/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Simbu virus/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
A new FFT-accelerated projection matching method is presented and tested. The electron microscopy images are represented by their Fourier-Bessel transforms and the 3D model by its expansion in spherical harmonics, or more specifically in terms of symmetry-adapted functions. The rotational and translational properties of these representations are used to quickly access all the possible 2D projections of the 3D model, which allow an exhaustive inspection of the whole five-dimensional domain of parameters associated to each particle.
Subject(s)
Cryoelectron Microscopy/methods , Image Processing, Computer-Assisted/methods , Fourier Analysis , Models, BiologicalABSTRACT
A method for finding the center of cryo-EM images which correspond to the projections of a symmetric 3D structure, based on mathematical properties of symmetry adapted functions and the Fourier-Bessel transform, is presented. It is a model independent one-step procedure with no parameters to be chosen by the user. The proposed method is tested in one synthetic tetrahedral case with different noise levels and in two real cases with D7 and icosahedral symmetries.
