Genome Organization of the Sigma Rhabdovirus: Six Genes and a Gene Overlap

The nonsegmented negative-strand RNA (NNS) viruses have a single-stranded RNA genome tightly encapsidated by the viral nucleocapsid protein. The viral polymerase transcribes the genome responding to specific gene-start and gene-end sequences to yield a series of discrete monocistronic mRNAs. These mRNAs are not produced in equimolar amounts; rather, their abundance reflects the position of the gene with respect to the single 3-proximal polymerase entry site. Promoter-proximal genes are transcribed in greater abundance than more distal genes due to a localized transcriptional attenuation at each gene junction. In recent years, the application of reverse genetics to the NNS viruses has allowed an examination of the role of the gene-start and gene-end sequences in regulating mRNA synthesis. These studies have defined specific sequences required for initiation, 5 modification, termination, and polyadenylation of the viral mRNAs. In the present report, working with Vesicular stomatitis virus, the prototypic Rhabdovirus, we demonstrate that a gene-end sequence must be positioned a minimal distance from a gene-start sequence for the polymerase to efficiently terminate transcription. Gene-end sequences were almost completely ignored in transcriptional units less than 51 nucleotides. Transcriptional units of 51 to 64 nucleotides allowed termination at the gene-end sequence, although the frequency with which polymerase failed to terminate and instead read through the gene-end sequence to generate a bicistronic transcript was enhanced compared to the observed 1 to 3% for wild-type viral mRNAs. In all instances, failure to terminate at the gene end prevented initiation at the downstream gene start site. In contrast to this size requirement, we show that the sequence between the gene-start and gene-end signals, or its potential to adopt an RNA secondary structure, had only a minor effect on the efficiency with which polymerase terminated transcription. We suggest three possible explanations for the failure of polymerase to terminate transcription in response to a gene-end sequence positioned close to a gene-start sequence which contribute to our emerging picture of the mechanism of transcriptional regulation in this group of viruses.

Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 77%

Identification of a Minimal Size Requirement for Termination of Vesicular Stomatitis Virus mRNA: Implications for the Mechanism of Transcription

Whelan

Barr

Wertz

2000

“…1A). While overlapping genes are known for other NNS RNA viruses, directly overlapping transcription signals are unique to filoviruses (16)(17)(18)(19)(20). Separating the overlapping VP35 GE and VP40 GS signals in an EBOV bicistronic minigenome (Bici) led to a decrease in downstream reporter activity and an increase in upstream reporter activity, indicating that the overlaps are of functional importance for EBOV gene expression (12).…”

Section: Importancementioning

confidence: 99%

Analysis of the Highly Diverse Gene Borders in Ebola Virus Reveals a Distinct Mechanism of Transcriptional Regulation

Brauburger

Boehmann

Tsuda

et al. 2014

Ebola virus (EBOV) belongs IMPORTANCEOur current understanding of EBOV transcription regulation is limited due to the requirement for high-containment conditions to study this highly pathogenic virus. EBOV is thought to share many mechanistic features with well-analyzed prototype nonsegmented negative-sense RNA viruses. A single polymerase entry site at the 3= end of the genome determines that transcription of the genes is mainly controlled by gene order and cis-acting signals found at the gene borders. Here, we examined the regulatory role of the structurally unique EBOV gene borders during viral transcription. Our data suggest that transcriptional regulation in EBOV is highly complex and differs from that in prototype viruses and further the understanding of this most fundamental process in the filovirus replication cycle. Moreover, our results with recombinant EBOVs suggest a novel role of the long IR found in all filovirus genomes during the viral replication cycle.

“…1D). Such a gene overlap in NNS RNA viruses has been found in the genera Pneumovirus (7,85), Ebolavirus (48), Bornavirus (56), and also the family Rhabdoviridae (45,64). Among these, a gene overlap in human respiratory syncytial virus (HRSV; a pneumovirus) appears to be most similar to that of CTRV in position and length; namely, a start signal of the HRSV L gene is located in the terminal tail of the upstream M2-1 gene, resulting in a 68-nt gene overlap.…”

Section: Discussionmentioning

confidence: 94%

RNA Splicing in a New Rhabdovirus from Culex Mosquitoes

Kuwata

Isawa

Hoshino

et al. 2011

Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.