Structure of Crimean-Congo hemorrhagic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage

Abstract

Crimean-Congo hemorrhagic fever, a severe hemorrhagic disease found throughout Africa, Europe, and Asia, is caused by the tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Nairovirus genus of the Bunyaviridae family. Its genome of three single-stranded RNA segments is encapsidated by the nucleocapsid protein (CCHFV N) to form the ribonucleoprotein complex. This ribonucleoprotein complex is required during replication and transcription of the viral genomic RNA. Here, we present the crystal structures of the CCHFV N in two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit with the base of the globular body of the adjacent subunit stabilizes the helical organization of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. By incubation with primer-length ssRNAs, we also obtained the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change of CCHFV N upon deoligomerization results in the exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. Thus, cleavage of CCHFV N by host cell caspase-3 appears to be crucial for controlling viral RNA synthesis and represents an important host defense mechanism against CCHFV infection.

Fig 1

(A) Crystal structure of CCHFV N. The structure is colored in rainbow colors from blue at the N terminus to red at the C terminus. (B) Superposition of 3 molecules of CCHFV N shows differences in the degree of bend of the stalk region. The three molecules, namely, A, B, and C, of CCHFV N in the asymmetric unit are colored in yellow, white, and cyan, respectively. (C) Head-to-tail oligomerization of the 3 molecules of CCHFV N in the asymmetric unit. The color scheme is the same as that in panel B.

Fig 2

(A) Antiparallel double superhelix polymer of CCHFV N. Each superhelix is colored in rainbow colors from blue to red. (B) Head-to-tail interactions of CCHFV N molecules at the caspase-3 cleavage site. One CCHFV N molecule is colored white. The adjacent CCHFV N involved in the head-to-tail interaction is colored yellow, with its residues that are identified as the caspase-3 cleavage site colored in magenta. (C) The caspase-3 cleavage site is obscured in the superhelical polymer. The caspase-3 cleavage site of CCHFV N in the superhelical form was superposed onto the structure of the inhibitor peptide bound to caspase-3 (PDB accession code 2CJX). The color scheme for CCHFV N is the same as that in panel B. The caspase-3 heterodimer is colored in cyan and blue, and the bound inhibitor peptide is colored in green.

Fig 3

Possible RNA-binding site of CCHFV N superhelical polymer. (A) Conserved clusters of positively charged residues that were identified are shown for part of the CCHFV N double superhelix. Yellow, cyan, and magenta surfaces depict the clusters composed of residues K132, R134, K135, and N468; residues H197, K222, R225, K282, K286, and K292; and residues K342, K343, and K346, respectively. (B) Electrostatic surface potential of the same part of the CCFHV-N double superhelix. Sulfate ions in the positively charged crevice are shown in spheres.

Fig 4

Conformational change of CCHFV N upon incubation with primer-length RNA. (A) Rotation of the stalk region upon incubation with primer-length RNA. The CCFHV N molecule in superhelical form is shown in white, and that in monomeric form is shown in red. (B) Conformational changes in the stalk domain. The color scheme is the same as that in panel A; only the stalk domains are shown for clarity. The arrow indicates the direction of movement. (C) Disruption of oligomerization by the conformational change. The color scheme is the same as that in panel A for the superposed CCHFV N molecules with neighboring CCHFV N molecules in the same helix colored in yellow and CCHFV N molecules in the juxtaposed antiparallel helix colored in black with a semitransparent surface. The arrow indicates the direction of movement. (D) The DEVD cleavage site is exposed when CCHFV N is incubated with primer-length RNA. Purified Ns were treated with or without poly(U)₁₂ ssRNA and incubated with human caspase-3. The reaction products were analyzed by SDS-PAGE followed by Western blotting. The pointers show full-length N (56 kDa) and the cleavage product (30 kDa). (E) The amount of cleaved N was measured by densitometry analysis from three independent experiments. The results are expressed as percentages of the cleavage product in reference to total N protein (cleaved and full-length N together).

Fig 5

Increase of CCHFV minigenome expression when the caspase recognition site DEVD of CCHFV N is mutated. BSR T7/5 cells were transfected with expression constructs for CCHFV polymerase (wild-type L_wt or inactive L_ΔDD), CCHFV nucleocapsid protein (wild-type N_blast or DEVD mutant N_D266+269A), CCHFV Gaussia luciferase minigenome (G-Luc), T7 polymerase, and firefly luciferase (FF-Luc) used as a transfection control. At 48 h after transfection, luciferase activities were determined, and counts were normalized to the negative control in which L_ΔDD is expressed. Mean values of G-Luc (black bars) and FF-Luc (gray bars) and standard errors of the means are shown (assays were triplicates, done twice). Significance is illustrated, with a t test P value of 0.027 (*).