RNA viruses are major global human and animal pathogens. Analysis of currently circulating viruses has been used to identify those most likely to cause future pandemics and these viruses should be the focus of an increased global effort to understand all aspects of their viral lifecycle. Recent analyses by the WHO and CEPI have identified Crimean-Congo Haemorrhagic fever virus (CCHFV) as one of these high priority viruses. CCHFV is a member of the Nairovirus family and is the cause of localised severe, high fatality human disease outbreaks.
The CCHFV genome is single stranded negative sense RNA that is segmented into three pieces and packaged into ribonucleoprotein complexes. In addition to the RNA, each ribonucleoprotein complex comprises many copies of the protective nucleoprotein and a single copy of the viral RNA-dependent RNA polymerase. The viral polymerase performs both replication (copying the genome) and transcription (producing viral mRNA). The polymerase of the CCHFV is almost twice the size of other RNA virus polymerase limiting inference from the few studies on related viruses. Despite this, the conservation of multiple enzymatic functions and several substrate binding domains makes the polymerase an ideal target for antiviral development.
To understand the function of the polymerase we first developed robust expression of the 4000 amino acid polypeptide and subsequently used cryoEM to determine several high-resolution structures. Our structures reveal the architecture of the core catalytic RNA -dependent RNA polymerase domain, revealing how genomic RNA is tethered inside the structure and identifying a large novel insertion above the polymerase core. Radionucleotide incorporation assays demonstrate the polymerase is highly active and of low fidelity, informing the potential development of nucleotide analogue inhibitors. Combined structural and functional analysis of the endonuclease reveal a highly potent enzyme with a non-canonical active site. The development of a large panel of nanobodies is being used to stabilise mobile domains and determine full length polymerase structure. Collectively these results provide the foundations for future work to unravel the molecular basis of CCHFV replication/transcription and for therapeutic development.