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Hazara nairovirus (HAZV) is a member of the Nairoviridae family within the Bunyavirales order, and closely-related to Crimean-Congo hemorrhagic fever virus that is responsible for severe and fatal human disease. The HAZV genome comprises three segments of negative sense RNA named S, M and L, with non-translated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis, and by analogy with other segmented negative sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3′ and 5′ NTRs exhibit extensive terminal complementarity; the first eleven nucleotides are strictly conserved and form promoter element (PE) 1, with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multi-step growth, which revealed the 3′ NTR was highly tolerant to change whereas several deletions of centrally-located nucleotides within the 5′ NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth, and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest its signalling ability relies solely on inter-terminal base pairing, and is an independent cis-acting signalling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed. |
