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The genomic, bipartite replication promoter of Ebola virus (EBOV) consists of elements 1 (PE1) and 2 (PE2). PE1 (55 nt at the 3′-terminus) is separated from PE2 (harboring eight 3′-UN 5  hexamers) by the transcription start sequence (TSS) of the first nucleoprotein (NP) gene plus a spacer sequence. Insertions or deletions in the spacer were reported to support genome replication if comprising 6 or 12, but not 1/2/3/5/9 nt. This gave rise to the formulation of the “rule of 6” for the EBOV replication promoter. Here, we studied the impact of such hexamer phasing on viral transcription using a series of replication-competent and -deficient monocistronic minigenomes, in which the spacer of the NP gene was mutated or replaced with that of internal EBOV genes and mutated variants thereof. Beyond reporter gene assays, we conducted qRT-PCR to determine the levels of mRNA, genomic and antigenomic RNA. We demonstrate that hexamer phasing is also essential for viral transcription, that UN 5  hexamer periodicity extends into PE1 and that the spacer region can be expanded by 48 nt without losses of transcriptional activity. Making the UN 5  hexamer phasing continuous between PE1 and PE2 enhanced the efficiency of transcription and replication. We show that the 2 nt preceding the TSS are essential for transcription. We further propose a role for UN 5  hexamer phasing in positioning NP during initiation of RNA synthesis, or in dissociation/reassociation of NP from the template RNA strand while threading the RNA through the active site of the elongating polymerase during replication and transcription.

Hexamer phasing governs transcription initiation in the 3′-leader of Ebola virus