Parainfluenza virus chimeric mini-replicons indicate a novel regulatory element in the leader promoter

et al. 2019

J Virol

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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.

Section: Methodsmentioning

confidence: 99%

“…The cDNA for the codon-optimized HAZV L ORF was synthesized (GeneArt, Ratisbonne, Germany) and cloned into the pTM1 vector (pTM1 HAZV L opt ). The firefly luciferase (Fluc) gene was cloned into the pTM1 vector as described previously (25). The nucleotide sequences of all the plasmids were confirmed by a DNA-sequencing method.…”

Section: Methodsmentioning

confidence: 99%

A Minigenome Study of Hazara Nairovirus Genomic Promoters

et al. 2019

J Virol

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“…Rluc expressing hPIV2 minigenome (hPIV2-Rluc) composed of leader, 5 ′ UTR of NP mRNA, 3 ′ UTR of L mRNA, trailer and Rluc gene, which has been used as "normal" hPIV2 minigenome (Matsumoto et al 2016(Matsumoto et al , 2017, was represented as AG/CRII minus in this study (hPIV2-Rluc AG/CRII minus ), because AG CRII that is intrinsically included in the coding region of L had been deleted. The hPIV2-Rluc with AG CRII (hPIV2 Rluc AG wt ) expressing plasmid was constructed by adding CRII to hPIV2-Rluc AG/CRII minus by using a standard PCR mutagenesis method.…”

Section: Plasmid Constructionmentioning

confidence: 99%

The control of paramyxovirus genome hexamer length and mRNA editing

et al. 2018

RNA

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The unusual ability of a human parainfluenza virus type 2 (hPIV2) nucleoprotein point mutation (NP) to strongly enhance minigenome replication was found to depend on the absence of a functional, internal element of the bipartite replication promoter (CRII). This point mutation allows relatively robust CRII-minus minigenome replication in a CRII-independent manner, under conditions in which NP is essentially inactive. The nature of the amino acid at position 202 apparently controls whether viral RNA-dependent RNA polymerase (vRdRp) can, or cannot, initiate RNA synthesis in a CRII-independent manner. By repressing genome synthesis when vRdRp cannot correctly interact with CRII, gln of N, the only residue of the RNA-binding groove that contacts a nucleotide base in the N-RNA, acts as a gatekeeper for wild-type (CRII-dependent) RNA synthesis. This ensures that only hexamer-length genomes are replicated, and that the critical hexamer phase of the -acting mRNA editing sequence is maintained.

“…Renilla luciferase expressing the minigenome plasmid of hPIV2 (hPIV2-Rluc) was constructed as described previously (14). Multiple luciferases, including Rluc, firefly luciferase (Fluc), and Gaussia secretory luciferase (Gluc), expressing minigenome plasmids of hPIV2 (hPIV2-Rluc-Fluc and hPIV2-Rluc-Fluc-Gluc) were constructed by a standard PCR method.…”

Section: Methodsmentioning

confidence: 99%

“…We have previously described hPIV2 minigenomes with successive 6-nt deletions in the leader region, which are inactive when NP wt supports minigenome replication (14). As they are all of hexamer length, their inactivity is presumably due to missing cis-acting sequences and/or because the spacing between the two elements of the bipartite promoter, which is critical, has been altered (15,16).…”

Section: Figmentioning

confidence: 99%

A Point Mutation in the RNA-Binding Domain of Human Parainfluenza Virus Type 2 Nucleoprotein Elicits Abnormally Enhanced Polymerase Activity

et al. 2017

J Virol

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The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as the template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA were identified and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (which contacts a base rather than the RNA backbone) to various amino acids resulted in an over 30-fold increase of polymerase activity as evidenced by a minireplicon assay, even though the RNA-binding affinity was unaltered. Using various modified minireplicons, we found that the enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA.IMPORTANCE We examined the importance of amino acids in the putative RNAbinding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed upon substitution mutation of the NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes that were partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for the infectious life cycle. This report highlights the potential of the polymerase complex with point mutations in NP and helps our detailed understanding of the molecular basis of gene expression. KEYWORDS human parainfluenza virus type 2, encapsidation, nucleoprotein, polymerase H uman parainfluenza virus type 2 (hPIV2) is a major respiratory pathogen and a member of the Rubulavirus genus of the family Paramyxoviridae, which includes simian virus 41, hPIV4, mumps virus, and PIV5. The hPIV2 genome RNA contains six tandemly linked genes (3=-NP-P/V-M-F-HN-L-5=) and is tightly coated by the nucleoprotein (NP) to form a helical nucleocapsid that serves as the template for viral RNA synthesis. The phosphoprotein (P) and large protein (L) together form the viral RNAdependent RNA polymerase (RdRp), which is responsible for both transcription to produce mRNAs and replication to produce genomes and antigenomes (1). hPIV2 NP contains 543 amino acids, whose functional domains are partially identified. The N-terminal 294 amino acids are required for NP self-assembly, and two C-terminal