Rescue of infectious recombinant hazara nairovirus from cDNA reveals the nucleocapsid protein DQVD caspase cleavage motif performs an essential role other than cleavage

J. Fuller, R. A. Surtees, G. S. Slack, J. Mankouri, Roger Hewson, J. N. Barr*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

Abstract

The Nairoviridae family of the Bunyavirales order comprises tick-borne, trisegmented, negative-strand RNA viruses, with several members being associated with serious or fatal diseases in humans and animals. A notable member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is the most widely distributed tick-borne pathogen and is associated with devastating human disease, with case fatality rates averaging 30%. Hazara virus (HAZV) is closely related to CCHFV, sharing the same serogroup and many structural, biochemical, and cellular properties. To improve understanding of HAZV and nairovirus multiplication cycles, we developed, for the first time, a rescue system permitting efficient recovery of infectious HAZV from cDNA. This system now allows reverse genetic analysis of nairoviruses without the need for high-level biosafety containment, as is required for CCHFV. We used this system to test the importance of a DQVD caspase cleavage site exposed on the apex of the HAZV nucleocapsid protein arm domain that is cleaved during HAZV infection, for which the equivalent DEVD sequence was recently shown to be important for CCHFV growth in tick but not mammalian cells. Infectious HAZV bearing an uncleavable DQVE sequence was rescued and exhibited growth parameters 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 role aside from caspase cleavage. IMPORTANCE HAZV is classified within the Nairoviridae family with CCHFV, which is one of the most lethal human pathogens in existence, requiring the highest biosafety level (BSL) containment (BSL4). In contrast, HAZV is not associated with human disease and thus can be studied using less-restrictive BSL2 protocols. Here, we report a system that is able to rescue HAZV from cDNAs, thus permitting reverse genetic interrogation of the HAZV replication cycle. We used this system to examine the role of a caspase cleavage site, DQVD, within the HAZV nucleocapsid protein that is also conserved in CCHFV. By engineering mutant viruses, we showed caspase cleavage at this site was not required for productive infection and this sequence performs a critical role in the virus life cycle aside from caspase cleavage. This system will accelerate nairovirus research due to its efficiency and utility under amenable BSL2 protocols.

Original languageEnglish
Article numbere00616-19
JournalJournal of Virology
Volume93
Issue number15
DOIs
Publication statusPublished - 2019

Bibliographical note

Funding Information:
We thank L. Bell-Sakyi and the Tick Cell Biobank, University of Liverpool (Liverpool, UK), for kindly providing HAE/CTVM9 cells. This work was funded by a Public Health England PhD studentship (to J.F.). J.F., R.A.S., G.S.S., J.M., R.H., and J.N.B. conceptualized the study, J.F., R.A.S., and G.S.S. performed the experimental investigation, J.F. and J.N.B. wrote the original draft manuscript, R.A.S. and G.S.S. reviewed and edited the manuscript, J.M., R.H., and J.N.B.

Funding Information:
We thank L. Bell-Sakyi and the Tick Cell Biobank, University of Liverpool (Liverpool, UK), for kindly providing HAE/CTVM9 cells. This work was funded by a Public Health England PhD studentship (to J.F.). J.F., R.A.S., G.S.S., J.M., R.H., and J.N.B. conceptualized the study, J.F., R.A.S., and G.S.S. performed the experimental investigation, J.F. and J.N.B. wrote the original draft manuscript, R.A.S. and G.S.S. reviewed and edited the manuscript, J.M., R.H., and J.N.B. supervised the core team, and J.N.B. provided management and coordination of the research activities and acquired the financial support for the project. We declare that there are no conflicts of interest.

Publisher Copyright:
© 2019 American Society for Microbiology. All Rights Reserved.

Keywords

  • Caspases
  • Hazara virus
  • Reverse genetics
  • Virus rescue

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