Structural Insight into Nucleoprotein Conformation Change Chaperoned by VP35 Peptide in Marburg Virus

Abstract

Marburg virus (MARV) encodes a nucleoprotein (NP) to encapsidate its genome by oligomerization and form a ribonucleoprotein complex (RNP). According to previous investigation on nonsegmented negative-sense RNA viruses (nsNSV), the newly synthesized NPs must be prevented from indiscriminately binding to noncognate RNAs. During the viral RNA synthesis process, the RNPs undergo a transition from an RNA-bound form to a template-free form, to open access for the interaction between the viral polymerase and the RNA template. In filoviruses, this transition is regulated by VP35 peptide and other viral components. To further understand the dynamic process of filovirus RNP formation, we report here the structure of MARV NP_core, both in the apo form and in the VP35 peptide-chaperoned form. These structures reveal a typical bilobed structure, with a positive-charged RNA binding groove between two lobes. In the apo form, the MARV NP exists in an interesting hexameric state formed by the hydrophobic interaction within the long helix of the NP_core C-terminal region, which shows high structural flexibility among filoviruses and may imply critical function during RNP formation. Moreover, the VP35 peptide-chaperoned NP_core remains in a monomeric state and completely loses its affinity for single-stranded RNA (ssRNA). The structural comparison reveals that the RNA binding groove undergoes a transition from closed state to open state, chaperoned by VP35 peptide, thus preventing the interaction for viral RNA. Our investigation provides considerable structural insight into the filovirus RNP working mechanism and may support the development of antiviral therapies targeting the RNP formation of filovirus.IMPORTANCE Marburg virus is one of the most dangerous viruses, with high morbidity and mortality. A recent outbreak in Angola in 2005 caused the deaths of 272 persons. NP is one of the most essential proteins, as it encapsidates and protects the whole virus genome simultaneously with self-assembly oligomerization. Here we report the structures of MARV NP_core in two different forms. In the MARV NP apo form, we identify an interesting hexamer formed by hydrophobic interaction within a long helix, which is highly conserved and flexible among filoviruses and may indicate its critical function during the virus RNP formation. Moreover, the structural comparison with the NP-VP35 peptide complex reveals a structural transition chaperoned by VP35, in which the RNA binding groove undergoes a transition from closed state to open state. Finally, we discussed the high conservation and critical role of the VP35 binding pocket and its potential use for therapeutic development.

Keywords: Marburg virus; assembly mechanism; crystal structure; filovirus; nucleoprotein.

FIG 1

Purification and crystal structure of MARV nucleoprotein core domain. (A) Construction of MARV nucleoprotein. Representative truncations are presented, including a full-length construct of NP_FL. The best truncation of MARV NP is NP_19–370 (NP_core). (B) Purification of MARV nucleoprotein. The sample containing MARV NP_core was injected into a Superdex-200 column for size exclusion chromatography (SEC). The molecular masses of standard protein markers are shown on the top. The blue and red lines indicate A₂₈₀ and A₂₆₀, respectively. SDS-PAGE analysis of the peak fractions is shown in the inset. (C) Cartoon representation of the overall structure of MARV NP_core. Missing residues are linked by dotted lines. Four positive residues, K142A, K153A, R156A, and K230 (shown as sticks in yellow), are labeled on NP. The protruding last α-helix (α20) is labeled. (D) EMSA of MARV NP_core WT and four mutations with ssRNA. Site-directed K142A, K153A, R156A, and K230A mutation proteins are sample loaded along with the wild type (WT) after incubation with an ssRNA probe. The dosages of NP and ssRNA probe are all set at 0.2 nM and 0.1 nM, respectively, as final concentrations. The free and combined probes are labeled. SDS-PAGE analysis of the MARV NP_core WT and mutation fractions are shown in the inset. The fraction binding for each mutation (black bars) is indicated in a chart made by GraphPad Prism after intensity quantification of the film by ImageJ and normalization to WT (gray bar). Error bars represent the standard deviations (SD) from three independent replicates.

FIG 2

Recombinant MARV NP_core exists as a hexamer both in crystals and in solution. (A) The hexamer of MARV NP_core in a cartoon representation (left) and in a 90°-rotated configuration (right). Three protomers representing a pair of the interactions inside the hexamer are colored blue, yellow, and purple, respectively. (B) A representative of the interactions inner the hexamer on α20 helices. The key residues are shown as sticks and labeled. The interactions between residues are depicted as dashed lines. (C) Mutational analysis of the interprotomer interface. SEC (Superdex-200 column) of MARV NP_core site-directed mutations I345D (red), I348D (purple), E350A (blue), I357D (orange), and F361D (green) are merged together with WT (gray) as the control.

FIG 3

MARV VP35 peptide chaperones NP_core. (A) MARV NP_core oligomerization is disrupted by VP35 peptide. SEC (Superdex-200 column) of MARV NP_core oligomer with VP35 peptide complex (red), merged together with MARV NP_core WT (blue). (B) ITC assay of the interaction between MARV NP_core and VP35 peptide. Data shown on the right side were calculated by Origin. Experiments were repeated more than three times. (C) Cartoon representation of MARV VP35 peptide rotating the C lobe of MARV NP_core. The conformation changes between the MARV NP_core apo form (blue) and its complex form (gray) are calculated by RMSD. The VP35 peptide (purple) induced a ∼25° lobe rotation, causing a potential opening of the RNA binding groove (blue bubble) and a potential RNA wrapping loop region (G310 to G319, dotted lines) movement. (D) Comparison of MARV and EBOV NP_core with or without VP35 peptides presented as a cartoon. The MARV NP_core apo form is colored in blue, and the EBOV NP apo form is colored in red. The two different EBOV NP (green for 4YPI and yellow for 4ZTG)-VP35 peptide (cyan for 4YPI and red for 4ZTG) complex structures are put into alignment with the MARV NP (gray)-VP35 peptide (purple) complex. The α20 and α21 helixes are labeled.

FIG 4

VP35 binds NP_core through hydrophobic interactions. (A) Sequence alignment of the conserved VP35 N-terminal peptide. The amino acids sharing identity are background-shaded red in blue squares, and similar amino acids are red in blue squares. The speculated potential key hydrophobic residues are indicated by arrowheads. (B) Representation of the hydrophobic interaction between MARV NP_core (surface in gray) and VP35 peptide (cartoon in purple). The potential key residues V10, S11, L14, and M15 are emphasized as sticks and colored in white. (C) Summary of ITC binding measurements between VP35 mutation derivatives and MARV NP_core. The experiments were conducted in the same manner as described for Fig. 3B, with at least three repeats.

FIG 5

The chaperon effect of VP35 on NP. (A) SDS-PAGE analysis of MARV NP_core (left lines) and NP_core-VP35 peptide complex (right lines) self-degradation. Each line contains the same quantity of original proteins. Time is given in hours (h) or days (d). The speculated region of MARV NP_core is boxed in red. Experiments were conducted in three individual repeats. (B) EMSA of the affinity competitions of VP35 peptide and ssRNA for NP. NP was pretreated with ssRNA and then incubated with VP35 peptide or in reverse order before sample loading. The dosages (final concentrations) are listed on the top. The free and combined ssRNAs are labeled on the side. Experiments were repeated more than three times. (C) Electrostatic surface potential (calcluated with APBS tools, with limits of ±5 kbT/ec) and cartoon representation for structure comparison of P/VP35 peptide or N^i-1 arm effect on NP (N). The P (green)/VP35 (pink) peptides, N^i-1 arms (orange), and α20 helices (cyan) are shown as a cartoon and labeled.