Molecular Insights into Crimean-Congo Hemorrhagic Fever Virus

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

In order to overcome some of the limitations inherent to the minigenome system, entry competent virus-like particle (VLP) systems have also been developed. A VLP contains all viral proteins and a minigenome. Therefore it is unable to express viral proteins upon entering a target cell. The particles in these systems can mimic a single-cycle infection; and because they do not encode CCHFV proteins, this system can be studied outside of high biocontainment laboratories. VLPs are generated by expressing three helper plasmids encoding the NP, GPC, and L protein together with a minigenome plasmid, resulting in the incorporation of encapsidated minigenome RNA into VLPs ( Figure 6) . The VLPs are able to enter target cells, and the RNPs can serve as templates for replication and transcription when the CCHFV NP and the L protein are supplied in trans. To date, two different CCHFV VLP systems have been developed [25] . In the first system, transcriptionally competent VLPs (tc-VLP) are incubated with target cells expressing the CCHFV NP and L protein in order to obtain robust Renilla reporter activity that can be used to study cell entry [25] . In the second system, transcriptionally and entry competent VLPs (tec-VLPs) are generated using a codon-optimized version of the L protein and GPC, and a NanoLuc reporter to produce VLPs. The resulting tec-VLPs can enter target cells to generate a robust NanoLuc signal without the need to express L protein and NP in the target cells, simplifying workflow for studying entry and primary transcription ( Figure 6 ) [27] . Figure 5 . CCHFV minigenome system. The CCHFV minigenome system is composed of a plasmid encoding the minigenome and three helper plasmids encoding the CCHFV NP, L, and T7 RNA polymerase (T7) genes downstream of a RNA polymerase II promoter. Downstream of a T7 promoter, the minigenome plasmid contains the 5′ and 3′ non-coding regions (NCR) of a CCHFV genomic segment (S, M, or L) flanking a gene encoding a reporter protein (NanoLuc) in the negative orientation. Transfection of the helper plasmids yields the corresponding proteins to enable transcription of the minigenome plasmid and production of minigenome-derived vRNA. Following T7 transcription, the vRNA is encapsidated to form the genomic RNP. The RNP is subsequently transcribed into mRNA (secondary transcription) and translated to yield the reporter protein or replicated to produce additional vRNA. vRNA generated by both T7 transcription and RNA replication can be used as templates for transcription of reporter gene mRNA by NP and the L protein, resulting in enhanced reporter activity. A measurable luminescent signal is produced by hydrolysis of an externally provided reporter substrate. Transfection of the helper plasmids yields the corresponding proteins to enable transcription of the minigenome plasmid and production of minigenome-derived vRNA. Following T7 transcription, the vRNA is encapsidated to form the genomic RNP. The RNP is subsequently transcribed into mRNA (secondary transcription) and translated to yield the reporter protein or replicated to produce additional vRNA. vRNA generated by both T7 transcription and RNA replication can be used as templates for transcription of reporter gene mRNA by NP and the L protein, resulting in enhanced reporter activity. A measurable luminescent signal is produced by hydrolysis of an externally provided reporter substrate.