Molecular Insights into Crimean-Congo Hemorrhagic Fever Virus

Zivcec, Marko; Scholte, Florine E. M.; Spiropoulou, Christina F.; Spengler, Jessica R.; Bergeron, Éric

The minigenome system is useful for studying viral transcription, replication, and encapsidation using model RNAs. Instead of a full-length vRNA or cRNA, it uses genome or antigenome analogues called minigenomes. The minigenome contains the terminal NCRs of a genomic segment, but the CCHFV coding region is replaced with a reporter gene [25] [26] [27] . Since the minigenome system is non-infectious and does not use full CCHFV genomes, it allows the study of CCHFV outside of high biocontainment laboratories. In the minigenome system, DNA copies of the vRNA minigenomes are cloned into expression vectors under the control of a T7 promoter; co-transfection with a T7-encoding plasmid yields a minigenome RNA ( Figure 5 ). As with other reverse genetics systems that rely on T7 promoter expression, a single terminal G is added to the 5 1 RNA termini to enhance T7 activity, and a native 3 1 terminus is generated using a hepatitis delta virus ribozyme [56] . The NP and L protein are provided either from expression plasmids or by superinfection with CCHFV. The minigenome RNA is encapsidated, and acts as a template for replication and for transcription, resulting in the production of mRNA and ultimately a reporter signal. The reporter signal provides a quantitative collective measure of genome replication, transcription and translation [112, 113] . However, by using an L protein with a mutated catalytic D693 it is possible to monitor replication alone as this mutant is unable to transcribe mRNA due to a lack of cap snatching activity [25] . While highly useful, the CCHFV minigenome system is limited as it does not model all aspects of the viral replication and cannot be used to study processes requiring the glycoproteins, such as entry, virus assembly, and egress.