Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro

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.

RNA Encapsulation Mode and Evolutionary Insights from the Crystal Structure of Emaravirus Nucleoprotein

“…The crystal structure of RNA-free N ΔN45 revealed four N ΔN45 molecules in the asymmetric unit that were organized in a tight tetramer, as was previously reported for N proteins from other members of the Bunyavirales order ( 14 , 18 , 21 , 22 ). Each of the monomers comprised an N-terminal arm (N arm, residues 52 to 71), a globular core (residues 71 to 280), and an α-helical C-terminal arm (C arm, residues 281 to 313) ( Fig.…”

The structure of a native orthobunyavirus ribonucleoprotein reveals a key role for viral RNA in maintaining its helical architecture

“…The N-and C-termini protrude from the core to form 'arms' that mediate homotypic oligomerisation, and this overall arrangement is shared by NPs from SBV, LACV and Leanyer orthobunyaviruses (11)(12)(13)(14)(15)(16). Orthobunyavirus NP crystallises as closed, non-physiological, tetrameric or hexameric rings (16) and this oligomeric promiscuity is confirmed by mass spectrometry and electron microscopy (EM), which reveal oligomers of increasing size from trimer to octamer (14,17,18).…”

The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture

“…The N- and C-termini protrude from the core to form ‘arms’ that mediate homotypic oligomerization, and this overall arrangement is shared by NPs from SBV, LACV, and Leanyer orthobunyaviruses ( 12 – 17 ). Recombinantly expressed orthobunyavirus NP crystallizes as closed, tetrameric, or hexameric rings ( 17 ) and this oligomeric promiscuity was confirmed by mass spectrometry and electron microscopy (EM), which revealed oligomers of increasing size from trimer to octamer ( 15 , 18 , 19 ).…”