Nairovirus nucleoproteins: development of diagnostic tools, structural characterisation and functional studies

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in case fatality rates of up to 60%, and for which no effective preventative or therapeutic measures are available. Here, the selection and biophysical characterisation of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV and possesses potential antiviral properties are described. Affimer-NP interferes in the RNA binding function of CCHFV NP, and inhibits CCHFV gene expression, reconstituted using a mini-genome system in mammalian cells. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for these interferences, with the Affimer-NP binding site positioned at a critical region of the NP involved in RNA binding and oligomerization. Additionally, the in vitro application of this novel molecule for the development of enzyme-linked immunosorbent and lateral flow assays is validated, presenting the first published pointof- care test able to detect CCHFV NP in spiked human and animal sera. The study of nairoviral NPs is further deepened using a reverse genetics system for Hazara virus (HAZV), a closely related homologue of CCHFV, for the incorporation of clonable tags to study the morphology and intracellular trafficking of nairoviral ribonucleoproteins (RNPs). A C-terminal 6xHis tag was engineered in the NP of HAZV for the purification of native viral RNPs in the context of infectious virus using affinity chromatography. Structural analyses of purified RNPs using electron microscopy revealed the helical structure of native nairoviral RNPs. A split-enhanced green fluorescent protein (split-eGFP) strategy was used to tag and visualise the NP of HAZV in infected cells. This system, combined with other fluorescence-based labelling techniques, allowed the characterisation of the intracellular dynamics of HAZV components and revealed the localisation of nairoviral replication factories using confocal microscopy. These results validate a novel approach to design and produce tagged-NP replication-competent recombinant HAZV that represents a valuable tool for the study of nairoviruses.