Structure, Function, and Evolution of the Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein

Abstract

c Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N-and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N-and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision.

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.…”

“…Interestingly, the structure described for the N protein of this strain revealed surfaces that may be implicated in RNA binding and N-N interactions. However, comparison of the crystal structure of the N protein of the Baghdad-12 strain with the crystal structure of the N protein of the YL04057 strain revealed differences in spatial positioning of domains (Carter et al, 2012;Guo et al, 2012). 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.…”

“…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.…”

“…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. Interestingly, the structure described for the N protein of this strain revealed surfaces that may be implicated in RNA binding and N-N interactions.…”

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.…”