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

Abstract

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.

Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on Global Gene Expression in Human Dendritic Cells

“…Even though the gradient of transcription is believed to be universal for nonsegmented negative-strand viruses (50), the levels of transcription of individual genes may not necessarily follow the gradient exactly due to the regulatory role of different elements of gene junctions, which may be different for different pairs of genes. The elements, which effect transcription, include intergenic regions and transcription termination signals, as demonstrated for respiratory syncytial virus (51,52) and recently for EBOV (53). In our study, the abundance of the eGFP mRNA was lower than that of the downstream genes at both time points.…”

Transcriptional Regulation in Ebola Virus: Effects of Gene Border Structure and Regulatory Elements on Gene Expression and Polymerase Scanning Behavior

“…Consequently, changes to the IR sequence or length have dramatic effects on gene expression in these viruses (reviewed in reference 1). In contrast, previous analyses with EBOV showed that the IRs are not essential for transcription of the adjacent genes (6,22). However, specific truncations of the 144-nt-long IR to 10, 20, or 30 nucleotides significantly inhibited downstream mRNA synthesis, while shorter or longer IRs did not result in substantial attenuation (6).…”

“…In contrast, previous analyses with EBOV showed that the IRs are not essential for transcription of the adjacent genes (6,22). However, specific truncations of the 144-nt-long IR to 10, 20, or 30 nucleotides significantly inhibited downstream mRNA synthesis, while shorter or longer IRs did not result in substantial attenuation (6). This finding is in contrast to reports for other viruses with variable IRs showing that either IR length did not influence downstream gene expression (23)(24)(25) or elongation of the IR proportionally reduced reinitiation efficiency (26).…”

“…The GS and GE signals that flank each EBOV gene are highly conserved (6)(7)(8)(9)(10). GS signals direct the viral polymerase to initiate mRNA synthesis and to cotranscriptionally cap and methylate the mRNA's 5= end, while the GE signals mediate polyadenylation and the termination of transcripts, as best characterized for vesicular stomatitis virus (VSV) and Sendai virus (11)(12)(13)(14)(15)(16).…”

“…1B, bottom). The GS of the downstream gene precedes the GE of the upstream one, and the overlap is restricted to a pentameric sequence motif, 3=-UAAUU, which is part of both the GE and GS signals (6,7,22). This makes filoviruses exceptional, as, to our knowledge, other NNS RNA viruses contain at most one overlapping gene border in which the much longer overlapping region is not limited to the transcription signals (27)(28)(29)(30)(31).…”