title: Transcriptional Regulation in Ebola Virus: Effects of Gene Border Structure and Regulatory Elements on Gene Expression and Polymerase Scanning Behavior.
abstract: The highly pathogenic Ebola virus (EBOV) has a nonsegmented negative-strand (NNS) RNA genome containing seven genes. The viral genes either are separated by intergenic regions (IRs) of variable length or overlap. The structure of the EBOV gene overlaps is conserved throughout all filovirus genomes and is distinct from that of the overlaps found in other NNS RNA viruses. Here, we analyzed how diverse gene borders and noncoding regions surrounding the gene borders influence transcript levels and govern polymerase behavior during viral transcription. Transcription of overlapping genes in EBOV bicistronic minigenomes followed the stop-start mechanism, similar to that followed by IR-containing gene borders. When the gene overlaps were extended, the EBOV polymerase was able to scan the template in an upstream direction. This polymerase feature seems to be generally conserved among NNS RNA virus polymerases. Analysis of IR-containing gene borders showed that the IR sequence plays only a minor role in transcription regulation. Changes in IR length were generally well tolerated, but specific IR lengths led to a strong decrease in downstream gene expression. Correlation analysis revealed that these effects were largely independent of the surrounding gene borders. Each EBOV gene contains exceptionally long untranslated regions (UTRs) flanking the open reading frame. Our data suggest that the UTRs adjacent to the gene borders are the main regulators of transcript levels. A highly complex interplay between the different cis-acting elements to modulate transcription was revealed for specific combinations of IRs and UTRs, emphasizing the importance of the noncoding regions in EBOV gene expression control.
  
  
  OBJECTIVE
  Our data extend those from previous analyses investigating the implication of noncoding regions at the EBOV gene borders for gene expression control. We show that EBOV transcription is regulated in a highly complex yet not easily predictable manner by a set of interacting cis-active elements. These findings are important not only for the design of recombinant filoviruses but also for the design of other replicon systems widely used as surrogate systems to study the filovirus replication cycle under low biosafety levels. Insights into the complex regulation of EBOV transcription conveyed by noncoding sequences will also help to interpret the importance of mutations that have been detected within these regions, including in isolates of the current outbreak.
authors.affiliation: Department of Microbiology, Boston University, Boston, Massachusetts, USA National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA Department of Virology, Philipps University of Marburg, Marburg, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Brauburger
authors.affiliation: Department of Virology, Philipps University of Marburg, Marburg, Germany.
authors.forename: Yannik
authors.initials: Y
authors.lastname: Boehmann
authors.affiliation: Department of Virology, Philipps University of Marburg, Marburg, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Krähling
authors.affiliation: Department of Microbiology, Boston University, Boston, Massachusetts, USA National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA muehlber@bu.edu.
authors.forename: Elke
authors.initials: E
authors.lastname: Mühlberger
mesh_terms: D000818:Animals; D002460:Cell Line; D021901:DNA, Intergenic; D029043:Ebolavirus / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D006801:Humans; D016366:Open Reading Frames; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D050436:Regulatory Elements, Transcriptional; D014158:Transcription, Genetic
chemical_list: D021901:DNA, Intergenic; D012324:RNA-Dependent RNA Polymerase