Reverse genetics system for emerging tick-borne orthonairovirus highlights dispensable N-terminal region of Gn associated with replication in tick vectors
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A growing number of emerging and re-emerging tick-borne orthonairoviruses belonging to the Sulina and Tamdy genogroups have recently been identified in association with febrile diseases in East Asia. Yezo virus (YEZV) is one such viruses and is genetically distinct from well- characterized Crimean-Congo hemorrhagic fever virus. Orthonairovirus glycoproteins (Gn and Gc) expressed from a single glycoprotein precursor (GPC) gene mediate interaction with host cells, yet the organization and functions of the YEZV glycoproteins remain largely undefined. To characterize YEZV glycoproteins in the context of infection, we developed a reverse genetics system that enables recovery of recombinant YEZV entirely from cloned cDNAs. The recombinant wild-type virus exhibited growth properties comparable to those of the parental isolate in vitro and maintained pathogenicity in vivo . Proteomic analysis of purified virus particles produced in mammalian cells showed that peptide coverage of GPC-derived products began at residue 69. We therefore used the reverse genetics system to examine the functional importance of the GPC subregion upstream of residue 69. A mutant with a deletion of GPC residues 28 to 68 (rYEZV GPCΔ28–68), which retained the predicted signal peptide, was successfully recovered, suggesting that this subregion is dispensable for producing infectious virus. rYEZV GPCΔ28–68 propagated at levels comparable to those of the wild-type virus in mammalian cells and exhibited similar pathogenicity in a mouse model. Interestingly, the mutant reached lower viral titers in tick-derived ISE6 cells and in ticks in vivo , suggesting that this N-terminal subregion may contribute to virus replication in ticks. Together, these findings demonstrate the utility of the newly established reverse genetics system for dissecting the functions of the YEZV glycoproteins in mammalian and tick systems.
Author Summary
Viral glycoproteins are important structural components on the surface of enveloped viruses and enable viruses to attach to and enter host cells. In orthonairoviruses, two glycoproteins Gn and Gc are produced from a single glycoprotein precursor (GPC) gene. However, the organization and functions of the glycoproteins of Yezo virus (YEZV), an emerging tick-borne orthonairovirus associated with febrile disease in East Asia, remain poorly understood. To characterize the YEZV glycoproteins, we developed a reverse genetics system for YEZV that allows us to engineer recombinant viruses. Using this platform, we found that a subregion spanning residues 28–68 of GPC was dispensable for recovery of infectious virus. In particular, the mutant grew at levels comparable to those of the wild-type virus in mammalian cells and caused similar disease progression in a mouse model but grew at lower levels in tick cells and in ticks. This contrast suggests that the deleted region may contribute to YEZV growth in tick systems. Together, our findings implicate a previously uncharacterized GPC region in host- dependent differences in YEZV replication and demonstrate the utility of reverse genetics for functional analysis of YEZV proteins.
