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