Hazara nairovirus requires COPI components in both Arf1-dependent and Arf1-independent stages of its replication cycle.

Hazara nairovirus (HAZV) is an enveloped tri-segmented negative strand RNA virus classified within the Nairoviridae family of the Bunyavirales order, and a member of the same subtype as Crimean-Congo hemorrhagic fever virus, responsible for fatal human disease. Nairoviral subversion of cellular trafficking pathways to permit viral entry, gene expression, assembly and egress is poorly understood. Here, we generated a recombinant HAZV expressing eGFP and used live-cell fluorescent imaging to screen an siRNA library targeting genes involved in cellular trafficking networks, the first such screen for a nairovirus. The screen revealed prominent roles for subunits of the coat protein 1 (COPI)-vesicle coatomer, which regulates retrograde trafficking of cargo between the Golgi and ER as well as intra-Golgi transport. We showed the requirement of COPI-coatomer subunits impacted at least two stages of the HAZV replication 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 1000-fold. Treatment of HAZV-infected cells with brefeldin-A (BFA), an inhibitor of Arf1 activation required for COPI coatomer formation, revealed this late COPI-dependent stage was Arf1-dependent, consistent with the established role of Arf1 in COPI vesicle formation. In contrast, the early COPI-dependent stage was Arf1-independent, with neither BFA treatment nor siRNA-mediated ARF1 knockdown affecting HAZV gene expression. HAZV exploitation of COPI components in a non-canonical Arf1-independent process suggests COPI coatomer components may perform roles unrelated to vesicle formation, adding further complexity to our understanding of cargo-mediated transport.IMPORTANCE Nairoviruses are tick-borne enveloped RNA viruses that include several pathogens responsible for fatal disease in humans and animals. Here, we analysed host genes involved in trafficking networks to examine their involvement in nairovirus replication. We revealed important roles for genes that express multiple components of the COPI complex, which regulates transport of Golgi-resident cargos. COPI components influenced at least two stages of the nairovirus replication cycle; an early stage prior to and including gene expression, and also a later stage during assembly of infectious virus, with COPI-knockdown reducing titres by approximately 1000-fold. Importantly, while the late stage was Arf1-dependent, as expected for canonical COPI vesicle formation, the early stage was found to be Arf1-independent, suggestive of a previously unreported function of COPI unrelated to vesicle formation. Collectively, these data improve our understanding of nairovirus host-pathogen interactions, and suggest a new Arf1-independent role for components of the COPI coatomer complex.