Mutagenic Analysis of Hazara Nairovirus Nontranslated Regions during Single- and Multistep Growth Identifies both Attenuating and Functionally Critical Sequences for Virus Replication

Abstract

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. IMPORTANCE Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3′ and 5′ ends of nairoviral genes, called non-translated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle such as mRNA transcription and RNA replication, with other roles likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3′ and 5′ NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.

A comprehensive list of the Bunyavirales replication promoters reveals a unique promoter structure in Nairoviridae differing from other virus families

“…3 A). Interestingly, this GC-rich dsRNA region is located after a spacer region composed of non-complementary bases around the 14th position in all three segments, as has been reported previously in HAZV and CCHFV 8 , 26 . We have previously suggested the possibility that the HAZV polymerase can recognize this GC-rich dsRNA as a promoter element essential for RNA synthesis initiation via an unidentified domain of the L protein 8 .…”

A comprehensive list of the replication promoters of Bunyavirales reveals a unique promoter structure in Nairoviridae differing from other virus families

“…The Nairoviridae promoter appears to have high G:C complementarity in the 17 to 21-nts region (Figure 2A and Supplementary Figure 2), and this likely reflects the high G:C complementarity rate at the promoter region (Figure 3A). Interestingly, this GC-rich dsRNA region is located after a spacer region composed of non-complementary bases around the 14th position in all three segments, as has been reported previously in HAZV and CCHFV (10,27). We have previously suggested the possibility that the HAZV polymerase can recognize this GC-rich dsRNA as a promoter element essential for RNA synthesis initiation via an unidentified domain of the L protein (10).…”