Characterization of the Ebola virus nucleoprotein-RNA complex

Abstract

When Ebola virus nucleoprotein (NP) is expressed in mammalian cells, it assembles into helical structures. Here, the recombinant NP helix purified from cells expressing NP was characterized biochemically and morphologically. We found that the recombinant NP helix is associated with non-viral RNA, which is not protected from RNase digestion and that the morphology of the helix changes depending on the environmental salt concentration. The N-terminal 450 aa residues of NP are sufficient for these properties. However, digestion of the NP-associated RNA eliminates the plasticity of the helix, suggesting that this RNA is an essential structural component of the helix, binding to individual NP molecules via the N-terminal 450 aa. These findings enhance our knowledge of Ebola virus assembly and understanding of the Ebola virus life cycle.

Fig. 1.

Characterization of the purified NP helix. (a) SDS-PAGE of the visible band isolated from the CsCl gradient. NP(Δ601–739) lacks C-terminal aa 601–739 of NP. NP(Δ451–739) lacks C-terminal aa 451–739 of NP. M, Molecular mass marker. (b) EM of negatively stained NP helices composed of wild-type NP, NP(Δ601–739) and NP(Δ451–739). Bars, 100 nm. A magnified area of the black rectangle is shown in each micrograph (b, insets). (c) Agarose gel electrophoresis of nucleic acids extracted from wild-type NP helices. The extracted nucleic acids were treated with mock, RNase A or DNase I. (d) Agarose gel electrophoresis of the RNA fraction extracted from wild-type NP helices after RNase A treatment.

Fig. 2.

EM of purified NP–RNA complex in different salt concentrations. Wild-type NP–RNA complex was dialysed against (a) 150 mM (b) 1 M (c) 0 mM NaCl in PB. Wild-type NP–RNA complex dialysed against 0 mM NaCl was redialysed against 150 mM NaCl (d). NP(Δ601–739)–RNA complex was dialysed against 0 mM NaCl in PB (e) and was redialysed against 150 mM NaCl (f). NP(Δ451–739)–RNA complex was dialysed against 0 mM NaCl in PB (g) and was redialysed against 150 mM NaCl (h). Bars, 100 nm.

Fig. 3.

EM of NP–RNA complex treated with RNase A. (a) NP–RNA complex in 150 mM NaCl in PB was treated with RNase A. (b) Sample (a) was then dialysed against 0 mM NaCl in PB. (c) NP–RNA complex in 0 mM NaCl in PB was treated with RNase A. (d) Sample (c) was then dialysed against 150 mM NaCl in PB. Bars, 100 nm.