Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage

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.

The Crimean-Congo Hemorrhagic Fever Virus NSm Protein Is Dispensable for Growth In Vitro and Disease in Ifnar-/- Mice

“…The NSm domain is not universally found in nairovirus genomes; NSm orthologs are only found in nairoviruses that phylogenetically cluster with CCHFV, including Nairobi sheep disease virus, Dugbe virus, Kupe virus, and Hazara virus. Until recently, investigating the role of nairovirus NSm proteins has been restricted by the absence of reverse genetics systems [15,16].…”

Hazara Nairovirus Requires COPI Components in both Arf1-Dependent and Arf1-Independent Stages of Its Replication Cycle

“…The expression strategy adopted involved the porcine teschovirus-1 2A peptide linker (P2A) sequence that induces ribosome skipping ( 22 – 24 ), to allow two open reading frames (ORFs) to be expressed from the HAZV S segment mRNA. The P2A linker was inserted between eGFP and HAZV N ORFs within previously described S segment expression plasmid (pMK-RQ-S) ( 21 ) to generate pMK-RQ-S(eGFP) ( Fig. 1A ).…”

“…Transfection of modified plasmid pMK-RQ-S(eGFP), along with wild-type (WT) M and L segment expression plasmids, was performed alongside a transfection comprising all three WT plasmids for comparison. An additional plasmid, pCAGGS-T7pol, which expresses bacteriophage T7 RNA polymerase was also cotransfected into cells, due to its previously described beneficial effects on the recovery of rHAZV (21,33). At 120 h posttransfection (hpt), supernatants were harvested and used to reinfect fresh monolayers of SW13s.…”

“…A detailed picture of how nairoviruses exploit cellular trafficking networks during all stages of the virus replication cycle have been limited by the paucity of virus-associated tools that permit large screening approaches. In this study, we used our recently developed reverse genetics system for HAZV ( 21 ) to rescue a recombinant HAZV expressing enhanced green fluorescent protein (rHAZV-eGFP) as a nonfused reporter protein, by inserting a P2A ribosome-skipping sequence from porcine teschovirus ( 22 – 24 ) within the HAZV S segment mRNA, as has recently been reported for CCHFV ( 25 ). We assessed the trafficking components required for rHAZV-eGFP infection using a targeted library of small interfering RNAs (siRNAs) specific for cellular components involved in vesicular transport and measured their influence on virus growth by live cell fluorescence microscopy.…”

Hazara virus and Crimean-Congo Hemorrhagic Fever Virus show a different pattern of entry in fully-polarized Caco-2 cell line

“…To get around the problem of BSL-4 laboratory requirement, Hazara virus (HAZV), a tickborne virus belonging to the same serogroup as CCHFV, has been proposed as a surrogate to CCHFV for in vitro and in vivo studies [15][16][17]. HAZV was isolated for the first time 1964 in Pakistan from Ixodes ticks [18], and there are no documented cases of human infections reported.…”