Regulation of VP30-Dependent Transcription by RNA Sequence and Structure in the Genomic Ebola Virus Promoter

Abstract

Viral transcription and replication of Ebola virus (EBOV) is balanced by transcription factor VP30, an RNA binding protein. An RNA hairpin at the transcription start site (TSS) of the first gene (NP hairpin) in the 3′-leader promoter is thought to mediate the VP30 dependency of transcription. Here, we investigated the constraints of VP30 dependency using a series of monocistronic minigenomes with sequence, structure and length deviations from the native NP hairpin. Hairpin stabilizations decreased while destabilizations increased transcription in the absence of VP30, but in all cases, transcription activity was higher in the presence versus absence of VP30. This also pertains to a mutant that is unable to form any RNA secondary structure at the TSS, demonstrating that the activity of VP30 is not simply determined by the capacity to form a hairpin structure at the TSS. Introduction of continuous 3′-UN5 hexamer phasing between promoter elements PE1 and PE2 by a single point mutation in the NP hairpin boosted VP30-independent transcription. Moreover, this point mutation, but also hairpin stabilizations, impaired the relative increase of replication in the absence of VP30. Our results suggest that the native NP hairpin is optimized for tight regulation by VP30 while avoiding an extent of hairpin stability that impairs viral transcription, as well as for enabling the switch from transcription to replication when VP30 is not part of the polymerase complex. IMPORTANCE A detailed understanding is lacking how the Ebola virus (EBOV) protein VP30 regulates activity of the viral polymerase complex. Here, we studied how RNA sequence, length and structure at the transcription start site (TSS) in the 3′-leader promoter influence the impact of VP30 on viral polymerase activity. We found that hairpin stabilizations tighten the VP30 dependency of transcription but reduce transcription efficiency and attenuate the switch to replication in the absence of VP30. Upon hairpin destabilization, VP30-independent transcription - already weakly detectable at the native promoter - increases, but never reaches the same extent as in the presence of VP30. We conclude that the native hairpin structure involving the TSS (i) establishes an optimal balance between efficient transcription and tight regulation by VP30, (ii) is linked to hexamer phasing in the promoter, and (iii) favors the switch to replication when VP30 is absent.

Identification and characterization of short leader and trailer RNAs synthesized by the Ebola virus RNA polymerase

“…As expected, mRNA+cRNA levels, predominantly representing mRNA levels ( Fig 4B ), largely decreased in the absence of VP30, while cRNA alone was significantly increased ( Fig 7A and 7B ). This is in line with previous qRT-PCR data demonstrating that a decrease of transcription correlates with an increase of replication [ 23 , 25 , 41 ]. In addition, leader RNA+cRNA levels were also increased in the absence of VP30 ( Fig 7C ).…”

“…The majority of termination sites coincide with the region of the NP hairpin (HP) structure. The NP HP is predicted to form on the genomic and/or antigenomic RNA level (nt 56–78, see Fig 1B ) and is involved in regulation of transcription by VP30 [ 26 , 41 , 44 , 45 ]. In the mRNA-enriched library, most NP mRNA reads had their 5’-end at the expected position 56 and minor fractions at nt 57 and 58 ( Fig 2B ).…”

“…The NP hairpin structure regulates VP30-dependent transcription initiation at the TSS [ 41 , 44 ]. To examine if VP30 affects leader RNA synthesis, we applied the qRT-PCR setup illustrated in S2B Fig .…”

“…However, RNA-Seq revealed that, even in the absence of VP30, mRNA synthesis was predominantly initiated opposite to genome nt -56 in case of the Δ5’ spacer MG ( Fig 8D and 8E ), the NheI NP MG ( S5 Fig ) and the wt (NP) MG ( S5 Fig ). Thus, recognition of the TSS and site-specific initiation of mRNA synthesis is basically the same in the presence or absence of VP30, although the overall efficiency of viral transcription is very low in the absence of VP30 [ 41 ].…”

“…MG variants were constructed as described [ 18 , 40 , 41 ], using Dpn I-based site-directed mutagenesis techniques (see S1 Fig ). Cloning primers are specified in S1 Table .…”