Development of an infectious clone system to study the life cycle of Hazara virus

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a negative sense single stranded RNA virus, capable of causing fatal hemorrhagic fever in humans. Currently, an infectious clone system exists for CCHFV, however, owing to the high containment level required for experimentation with this virus, its potential is limited. Here, a highly efficient infectious clone system was developed for recovery of Hazara virus (HAZV), the first such orthonairovirus system able to be used in biosafety level 2 facilities, providing a valuable tool for increasing our understanding of these viruses. Mechanisms of viral subversion of cellular trafficking pathways involved in viral entry, gene expression, assembly and egress are poorly understood for HAZV and CCHFV. The infectious clone system was adapted to express eGFP, enabling screening of an siRNA library targeting genes involved in cellular trafficking networks via live-cell fluorescent imaging, the first such screen for a nairovirus. Screening revealed an important role for subunits of the coat protein 1 vesicle coatomer (COPI), normally involved in regulation of Golgi / ER cargo trafficking. The effect was observed at multiple stages of the HAZV life cycle; an early stage prior to and including gene expression, and also a later stage during assembly and egress of infectious virus, with COPI-knockdown reducing titres by approximately 100-fold. In addition to gain of function mutations, such as the reporter virus discussed above, the infectious clone system represents a powerful tool for exploring the role of individual nucleotides, amino acids and entire open reading frames within the viral genome. Both CCHFV and HAZV contain a well-documented, highly conserved caspase cleavage motif on the apex of the arm domain on the nucleoprotein (N). Whilst previous literature has demonstrated this motif to be cleaved as purified protein, limited data exists as to its role in the context of viral replication in a cellular system. To this end, the infectious clone system described above was used to create a panel of mutants targeting this conserved caspase cleavage motifs within HAZV N. HAZV bearing an uncleavable DQVE sequence rescued efficiently with growth rates equivalent to those of wild-type virus in both mammalian and tick cells, showing this site was dispensable for virus multiplication. In contrast, substitution of the DQVD motif with the similarly uncleavable AQVA sequence could not be rescued despite repeated efforts. Together, these results highlight the importance of this caspase cleavage site in the HAZV life cycle but reveal the DQVD sequence performs a critical, as yet unknown, role aside from caspase cleavage.