Structure of Crimean-Congo Hemorrhagic Fever Virus Nucleoprotein: Superhelical Homo-Oligomers and the Role of Caspase-3 Cleavage

Abstract

h 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.

Mapping of the interaction domains of the Crimean–Congo hemorrhagic fever virus nucleocapsid protein

“…These functions are mediated by multiple interactions with viral and cellular factors. Recently, three independent groups reported the crystal structure of the CCHFV N protein (Carter et al, 2012;Guo et al, 2012;Wang et al, 2012). These crystal structures provided hints on the spatial positions of structural features of the N protein, and enabled studies on structure-function relationships, including its oligomerization and RNA-binding domains.…”

“…3b). The mutants were designed to contain different structural regions according to the structure published by Wang et al (2012). This structure revealed two domains in the N protein: the N-terminal residues 1-183 and the C-terminal residues 295-482 combine to form a globular head domain, while residues 195-294 form a stalk protruding from the globular head.…”

“…In addition, Guo et al (2012) showed that the monomeric form of the CCHFV N protein has low RNA-binding affinity but a strong metaldependent DNA-endonuclease activity. Wang et al (2012) reported that the N protein of the IbAr10200 strain possesses two different conformations: one resembling an oligomerized RNP and a monomeric form with an exposed caspase-3 cleavage site. Furthermore, the disruption of the caspase-3 cleavage site resulted in an enhancement of the CCHFV polymerase activity during vRNA transcription.…”

“…Recently, three independent groups (Carter et al, 2012;Guo et al, 2012;Wang et al, 2012) have determined the crystal structure of the N protein of CCHFV, unravelling critical information about its structure-function relationships and elucidating intramolecular interactions. Carter et al (2012) showed that the N protein monomer of the Baghdad-12 strain resembles the structure of the nucleoprotein of Lassa virus, a member of the family Arenaviridae, suggesting that nairoviruses and arenaviruses might share a common ancestor.…”

Expression, purification, crystallization and preliminary X-ray crystallographic study of the nucleocapsid protein ofTomato spotted wilt virus

“…Recently, the structures of N proteins from three animal virus genera of the Bunyaviridae family have been reported and several encapsidation mechanisms of viral genome RNAs have been suggested based on the formation of different oligomers of N proteins (Raymond et al, 2010(Raymond et al, , 2012Ferron et al, 2011;Guo et al, 2012;Carter et al, 2012;Wang et al, 2012;Dong et al, 2013;Niu et al, 2013;Li et al, 2013;Reguera et al, 2013). Interestingly, the sequence and structure of N proteins are highly conserved within a genus but differ between genera.…”