Structural and cellular biology of the nairovirus nucleocapsid protein

Crimean Congo haemorrhagic fever virus (CCHFV) is a tick borne nairovirus responsible for a highly fatal human disease with increased recent incidence within the human population of southern Europe. Hazara virus (HAZV), another tick borne nairovirus of the same serogroup as CCHFV, is not pathogenic in humans, but infection with HAZV results in a similar disease progression in interferon receptor knockout mice to CCHFV. In order to investigate further similarities between HAZV and CCHFV, and to support the use of HAZV as a model for CCHFV infection, various structural and cell biological aspects of the CCHFV and HAZV replication cycle were investigated. We present the X-ray crystal structure of the nucleocapsid protein (N) of HAZV at a resolution of 2.7 Å, and the crystal structure of RNA-bound HAZV N at a resolution of 3 Å. HAZV N had a highly similar overall structure to CCHFV N, consisting of a globular domain containing amino acid residues from both the N and the C-termini, and an extended arm domain. A crevice within the globular domain of HAZV N had 3 nucleotides of RNA bound, in the RNA-bound HAZV N structure. RNA binding is a crucial function of HAZV N and CCHFV N, and the identification of an RNA binding site in HAZV N provides a model for the structure guided design of anti-virals to disrupt this interaction. Cellular heat shock protein 70 (HSP70) was found to be an interacting partner of the N protein of CCHFV and HAZV, both in cells and in secreted HAZV particles. The functional role of this interacting partner within the HAZV replication cycle was investigated using siRNA knock down and small molecule inhibitors of endogenous HSP70. HAZV induced apoptosis in SW13 cells, and HAZV N was cleaved during a 72 hr time course of infection. The data presented here also reveals new insights into the replication cycles of HAZV and CCHFV