Expession, Purification, Crystallization, Structural And Fuctional Analysis Of CCHFV Nucleoprotein

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne and a severe human disease with high case fatality (30%-50%).The disease is widespread all over the world, it has been reported in30countries in Africa, Asia, Europe, the Middle East. In1965, it first surfaced at Bachu county, xinjiang China (also named as XHF) and resulted in high fatality (～80%). CCHF is primarily prevailing in Southwest China and Northwest China. People infect this disease through two ways, one way is direct infection by tick, another way is indirect infection by skin lesions. The main symptoms of CCHF include high fever, severe headache, nausea, vomiting, hemorrhagic manifestations, liver and renal failure. Currently there is no vaccine for the disease and there are no signs of effective drug treatments. The causative agent of Crimean-Congo hemorrhagic fever is CCHFV, a negative-sense single-stranded RNA virus which belongs to the genus Nairovirus within the family Bunyaviridae. The genome is consisted of three RNA segment L, M, S, encoding L polymerase, Gn and Gc glycol-proteins, N nucleocapsid. Because of its strong infectious, CCHFV is a potential bioterrorism agent, and is classified as a biosafety level4pathogen. Until now, the basic research about CCHFV is limited.The three single-stranded RNA segments of CCHFV genome is encapsidated by the nucleocapsid protein to form the ribonucleoprotein complex (RNP), which is required for both RNA replication, transcription and assembly of virion. It also protects RNA from host cell degradation. NP can interact with viral proteins and host proteins, in order to disturb the normal function of the host.In this work, our study focuses on the nucleoprotein of CCHFV, and we do a lot of experiments including the expession, purification, crystallization of CCHFV NP and so on. A cDNA encoding fragment of N was inserted into a vector, we expressed CCHFN NP in Eloli cells, purificated and crystalled. AnNP data set was collected to a resolution of2.3A. Unlike others, CCHFV NP is composed of two distinct domain,"head" domain and "stalk" domain, like a racket. we also do series of biochemical experiments to research the function of CCHFV NP, including the status of CCHFV NP, binding affinity with RNA, interaction with caspase3, DNA-specific endonuclease activity.Structural and functional studies on CCHFV NP will provide direct and accurate proof for the instruction of virus genome assembling and mRNA transcription mechanism. It also provides a true drug target for the prevention and treatment of CCHF.