A Minigenome Study of Hazara Nairovirus Genomic Promoters

Abstract

Hazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the order Bunyavirales. The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis. IMPORTANCE A minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3′ and 5′ strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

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

“…The results of the previous section showed that mutants 3'D6 and 3'D7 were viable but extremely unfit, and mutants 5'D12 and 5'D13 could not be rescued. Interestingly, for mutants 3'D7, 5'D12 and 5'D13, the altered nucleotides fall within PE2, the terminal-distal promoter region found to require, in major part, inter-terminal Watson-Crick base pairing for its promoter activity (25). The number of potential complementary interterminal parings within these three mutants differs; for 3'D7 it is just two (Fig 2H) PE2 represents an independent and modular cis-acting sequence signal.…”

“…A total of seven 3' NTR deletions and thirteen 5' NTR deletions were individually engineered into the corresponding S segment plasmid (Fig 1C), with the changes spanning the entire 3' and 5' NTRs and including previously described PE1 and PE2 (25). Plasmids were named 3'D1 to 3'D7 and 5'D1 to 5'D13, accordingly (Fig 1C), and the full 3' and 5' NTR sequence of all corresponding mutants is shown in Figure S1A and S1B, respectively.…”

“…A first and recent study to define nairovirus NTR functionality employed HAZV mini-genomes to describe promoter elements (PEs) that were involved in the signalling of reporter activity as a marker for RNA synthesis (25); PE1 comprised strictly conserved 3' and 5' terminal-proximal sequences, whereas PE2 comprised a GC-rich sequence that was predicted to form inter-terminal Watson-Crick pairings, similar to that described previously for BUNV (10,20). PE1 and PE2 were found to be separated by a 'spacer' region, which exhibited a critical requirement for short length and lack of base-pairing ability (Fig 1B).…”

The Cap-Snatching Mechanism of Bunyaviruses

“…However, this cannot be the only strategy in place as the Z protein is only encoded by members of the Arenaviridae with no obvious counterpart in other bunyaviruses. Most importantly, bunyavirus transcription has been proven to be solely dependent on RNP components [54][55][56][57][58].…”

Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage

“…The development of a HAZV rescue system that can be utilized using less-restrictive BSL2 protocols will greatly facilitate and accelerate future nairovirus research. A HAZV minigenome system was also reported recently (29), which allowed the delineation and characterization of the viral mRNA transcription promoters, and, while the utility of this system is restricted to analysis of cis-acting signals involved in RNA synthesis, it will also allow a rapid accumulation of information regarding nairovirus multiplication.…”