Insight into the Ebola virus nucleocapsid assembly mechanism: crystal structure of Ebola virus nucleoprotein core domain at 1.8 Å resolution

Abstract

Ebola virus (EBOV) is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA (-ssRNA) viruses, EBOV possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex (RNP) together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain (NP(core)) possesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an α-helix of EBOV NP(core) itself, which is highly conserved among filoviridae family. Combined with other biochemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.

Figure 1

Purification and structure of EBOV NP _core. (A) Size exclusion chromatography (SEC) of EBOV NP_core. The sample containing EBOV NP_core was injected into a Superdex-200 column. The molecular weights of standard protein markers are shown on the top. Blue and red lines denote A₂₈₀ and A₂₆₀, respectively. SDS-PAGE analysis of the peak fractions is shown inset. (B) Overall structure of EBOV NP_core. The polypeptide of EBOV NP_core is shown as colored cartoon. The N-lobe and C-lobe are colored as blue and red, respectively. Missing residues are linked by dotted lines. (C) Schematic diagram of the domain organization in the primary sequence of EBOV NP. N-lobe, C-lobe, and C-tail are colored as slate blue, salmon red, and green, respectively. N-tail and non-conservative region are colored as blank. (D) Topology diagram of EBOV NP_core helices are presented as rectangulars and strands are shown as arrows. The color scheme is the same as that in (B) and (C)

Figure 2

Potential RNA binding region of EBOV NP _core. (A) Cartoon representation of EBOV NP_core. The N-lobe and C-lobe are colored as slate blue and salmon red, respectively. (B) Electrostatic surface potential of EBOV NP_core. The electrostatic surface potential of EBOV NP_core was calculated using APBS tools, with limits ±5 kbT/ec. Positive residues are highlighted by red circle on EBOV NP_core, suggesting the presence of several positively charged grooves that may be involved in RNA binding

Figure 3

Structural comparison of NPs from Mononegavirales viruses. (A) Structural comparison of EBOV NP_core with RSV, PIV5, and NiV NP. NPs are displayed as cartoon. RSV (PDB code: 2WJ8), PIV5 (PDB code: 4XJN), and NiV (PDB code: 4CO6) NPs are colored cyan, gray, and yellow, respectively. Alignment information is listed under each molecule. All molecules are aligned to the structure of EBOV NP_core and shown in the same orientation. (B) Electrostatic potential comparison of EBOV NP_core with RSV, PIV5, and NiV NP. Positively charged pockets for RNA binding are indicated with orange arrows. RNA molecules in RSV and PIV5 NPs are shown as colored sticks

Figure 4

Helix-20 in EBOV NP _core indicates an essential hydrophobic pocket. (A) Comparison of EBOV NP_core and NiV NP. Both structures are shown as surface and colored gray. EBOV NP_core Helix-20 and NiV P₅₀ are shown as cartoon, colored salmon red and cyan, respectively. (B) Formation of the hydrophobic pocket. Main chain is shown in cartoon and hydrophobic residues are shown in stick, colored in red by hydrophobicity. (C) Interaction of Helix-20 with hydrophobic pocket. Hydrophobic pocket are shown in surface, Helix-20 are displayed in ribbon and stick, colored in red by hydrophobicity. (D) Primary sequence alignment of members of the filoviridae family. Highly conserved residue in Helix-14, Helix-15, Helix-19, and Helix-20 are indicated by yellow arrow

Figure 5

Structural mobility of EBOV NP _core within virus proliferation. (A) Structural comparison of EBOV NP_core and complex with VP35 peptide. The molecule of EBOV NP_core alone and in complex with VP35 peptide are shown in the left and right panels. The N-lobe and C-lobe are colored as blue and red, respectively. Helices 20–21 are shown as cylinder, and the loop region (A326–V334) are linked by dotted lines. Small peptide derived from VP35 is shown as yellow cylinders. (B) A proposed model of conformation change during nucleocapsid assembly and transcription process. RNA binding groove and hydrophobic pocket are indicated by arrow