The Bunyamwera Virus mRNA Transcription Signal Resides within both the 3′ and the 5′ Terminal Regions and Allows Ambisense Transcription from a Model RNA Segment

Abstract

Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of RNA viruses. BUNV genomic strands are templates for both replication and transcription, whereas the antigenomic RNAs serve only as templates for replication. By mutagenesis of model templates, we showed that the BUNV transcription promoter comprises elements within both the 3 and the 5 nontranslated regions. Using this information, we designed a model ambisense BUNV segment that transcribed BUNV S mRNA from the genomic strand and green fluorescent protein (GFP) mRNA from the antigenome. Demonstration of GFP expression showed that this ambisense strategy represents a viable approach for generating BUNV segments able to express additional proteins.The Bunyaviridae family of RNA viruses comprises five genera, namely, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. Many bunyaviruses cause life-threatening human disease and consequently are described by the Centers for Disease Control and Prevention as category A, B, and C priority pathogens. The prototype of the Bunyaviridae family is Bunyamwera virus (BUNV), which serves as a model for the many pathogens within this family.The BUNV genome comprises three segments of negativesense RNA, designated small (S), medium (M), and large (L). The S segment encodes the nucleocapsid (N) and nonstructural proteins (NSs) expressed from overlapping open reading frames (ORFs) on the same mRNA (8,11,12). The M segment encodes a polyprotein that is cleaved to yield Gn, Gc, and NSm proteins (6,12,13). The L segment encodes the RNA-dependent RNA polymerase (RdRp) (7).The ORFs of each segment are flanked by nontranslated regions (NTRs) that direct the RdRp to perform two distinct RNA synthesis activities: (i) transcription to generate a single mRNA and (ii) replication to generate antigenomes that are replicated to generate further genomes. The BUNV S-segment genome is a template for both replication and transcription, whereas the antigenome serves only as a template for replication (5,14). This division of template activity, in which a functional mRNA is transcribed only from the genomic strand, is not a universal feature of all bunyavirus members. Many viruses within this group perform ambisense transcription, in which both genome and antigenomes are transcriptionally active. The ambisense strategy of gene expression is a feature of the segmented arenaviruses and recently has been artificially bestowed upon several nonsegmented negative-sense RNA viruses (9, 18).The fundamental difference in activity of the BUNV genomic and antigenomic RNAs suggests that these strands are recognized differently by the BUNV RdRp, which is likely due to critical nucleotide differences within the 3Ј and 5Ј NTRs of these RNAs. For the BUNV S segment, the 3Ј and 5Ј NTRs are 85 and 170 nucleotides in length, respectively. To facilitate our search for nucleotides responsible for the different strand activities, we constructed WT(25/25), which comprises nucleotides 1 to 25 of 3Ј and 5Ј NTRs surrounding the intact BUN...

Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus

“…The parental construct for these deletions was the previously described model segment 25/25 (Barr et al 2005), which contained the first 25 nt of both 39 and 59 S segment NTRs, and which replicated with an ability approaching that of the wild type. A total of five model segments having deletions within their anti-genomic 39 ends were constructed and were designated 25/23, 25/21, 25/18, and 25/17, with the numbers corresponding to the lengths of the 59 and 39 anti-genomic NTRs, respectively.…”

“…Therefore, when we aligned the 59 bound RNA sequences, we also included the BUNV 59 NTRs in the analysis. Furthermore, as our previous work showed that just 25 nt at the 59 end of the S segment is sufficient for replication and thus encapsidation of either BUNV genomic or anti-genomic strands (Barr et al 2005), we included only these nucleotides in the alignment. Our analysis failed to detect any common motifs within these sequences, and thus suggested that the bound RNAs were unlikely to be encapsidated by virtue of a specific signal formed by a linear nucleotide sequence.…”

“…This plasmid was subsequently modified to generate plasmid p25/25, which expressed a model segment containing the first 25 nt of 59 and 39 NTRs of the S segment anti-genomic strand (Barr et al 2005). Further truncations were made such that nucleotides were progressively removed from the terminal-distal side of the NTRs.…”

Mutational Analysis of the Lassa Virus Promoter

“…Relevant examples are the promoter models for influenza A virus (2, 6-8, 10, 11, 15, 16, 22, 24, 26), Thogoto virus (19,20), Bunyamwera virus (3,4,17), and Uukuniemi virus (9). Great similarity in structure and function is seen between the Lassa virus, Uukuniemi virus, and Bunyamwera virus promoters.…”

“…Deletion analysis showed that both 3Ј and 5Ј termini are required for transcriptional activation, indicating that the promoter indeed functions as a duplex. The promoter elements of several segmented negative-strand RNA viruses, including influenza A virus (2, 6-8, 10, 11, 15, 16, 22, 24, 26), Thogoto virus (19,20), Bunyamwera virus (3,4,17), and Uukuniemi virus (9), have already been characterized in detail, and structural models of the promoter have been proposed. A detailed analysis of the arenavirus promoter is still lacking.…”