RNP Research Papers - Academia.edu (original) (raw)

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. C rimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic virus responsible for a serious human disease characterized by hemorrhagic manifestations and multiple organ failure and is associated with a fatality rate of up to 50% (13, 46). The geographic range of CCHFV is exceptionally wide and reflects the broad distribution of the tick vector, which extends throughout 30 countries within Africa, Asia, the Middle East, and Southern Europe (25). Recent outbreaks of CCHFV infection in several Balkan states, southwestern Russia, and Turkey suggest that the activity of CCHFV is increasing, particularly in Southern Europe (24). The only treatment for CCHFV infection is postexposure administration of ribavirin, and the efficacy of this prophylaxis is in doubt (45). Development of effective treatments for prevention of CCHFV-mediated disease is now a priority for both public health and biodefense agencies. CCHFV is a member of the Bunyaviridae family, and together with members of the Arenaviridae and Orthomyxoviridae families, these viruses are known as segmented negative-strand RNA viruses (sNSVs) by virtue of their multiple genome segments. The Bunyaviridae family contains over 350 named isolates classified within five genera, namely, Hantavirus, Nairovirus, Orthobunya-virus, Phlebovirus, and Tospovirus (41). CCHFV is a nairovirus, with a genome comprising three RNA segments: the small (S), medium (M), and large (L) segments. The S segment encodes the nucleocapsid protein (N), the M segment encodes the viral glyco-proteins, and the L segment encodes an RNA-dependent RNA polymerase (RdRp; the L protein). The genomes of sNSVs do not exist as naked RNAs but instead are encapsidated by the viral N protein to form ribonucleoprotein (RNP) complexes. RNPs associate with their cognate RdRp to form active templates for viral RNA synthesis, resulting in generation of encapsidated replication products and unencapsidated mRNAs. Genome encapsidation is also required for RNP packaging into progeny virus particles, and for bunyaviruses, virus assembly is mediated through direct association between the RNP and viral glycoproteins (18, 31, 39, 42, 44). RNP formation is thus essential for virus multiplication and therefore represents a potential therapeutic target. In addition to RNP formation and virus assembly, the N proteins of bunyaviruses are implicated in other important functions, many of which relate to interactions with components of the host cell, including the cytoskeleton (3, 35–37, 43), cellular RNAs (26, 28), the translation machinery (8, 27), and mediators of the innate immune response (20, 30). Specifically for CCHFV, the N protein interacts with the cellular antiviral defense factor MxA (2) and recently was shown to act as a substrate for the apoptosis mediator caspase-3 (19), although the relevance of caspase-3 cleavage to the virus life cycle is unknown. We present the 2.1-Å crystal structure of the N protein from CCHFV strain Baghdad-12. The CCHFV N structure we present displays significant differences in domain position compared to the recently reported structure of N from CCHFV strain YL04057