[Source: Journal of Virology, full page: (LINK). Abstract, edited.]
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, R. Hewson, J. N. Barr
The Nairoviridae family of the Bunyavirales order comprises tick-borne tri-segmented negative strand RNA viruses, with several members associated with serious or fatal disease in humans and animals. A notable member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is the most widely-distributed tick-borne pathogen, and 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 genetics analysis of nairoviruses without the need for high 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, and 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 un-cleavable DQVE sequence was rescued and exhibited equivalent growth parameters to wild-type in both mammalian and tick cells, showing this site was dispensable for virus multiplication. In contrast, substitution of the DQVD motif with the similarly un-cleavable AQVA sequence could not be rescued despite repeated efforts. Together, this work highlights the importance of this caspase cleavage site in the HAZV lifecycle, but reveals the DQVD sequence performs a critical role aside from caspase cleavage.
Hazara virus is classified within the Nairoviridae family along with Crimean-Congo hemorrhagic fever virus (CCHFV), which is one of the most lethal human pathogens in existence, requiring the highest biosafety level (BSL) containment (BSL-4). In contrast, HAZV is not associated with human disease and thus can be studied using less-restrictive BSL-2 protocols. Here, we report a system able to rescue Hazara virus (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 furthermore that this sequence performs a critical role in the virus lifecycle aside from caspase cleavage. This system will accelerate nairovirus research due to its efficiency and utility under amenable BSL-2 protocols.
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Keywords: Bunyavirus; Hazara virus; CCHF virus; Nairovirus.