ABSTRACT

Ebola virus (EBOV) RNA has the potential to form hairpin structures at the transcription start sequence (TSS) and reinitiation sites of internal genes, both on the genomic and antigenomic/mRNA level. Hairpin formation involving the TSS and the spacer sequence between promotor elements (PE) 1 and 2 was suggested to regulate viral transcription. Here, we provide evidence that such RNA structures form during RNA synthesis by the viral polymerase and affect its activity. This was analysed using monocistronic minigenomes carrying hairpin structure variants in the TSS-spacer region that differ in length and stability. Transcription and replication were measured via reporter activity and by qRT-PCR quantification of the distinct viral RNA species. We demonstrate that viral RNA synthesis is remarkably tolerant to spacer extensions of up to ~54 nt, but declines beyond this length limit (~25% residual activity for a 66-nt extension). Minor incremental stabilizations of hairpin structures in the TSS-spacer region and on the mRNA/antigenomic level were found to rapidly abolish viral polymerase activity, which may be exploited for antisense strategies to inhibit viral RNA synthesis. Finally, balanced viral transcription and replication can still occur when any RNA structure formation potential at the TSS is eliminated, provided that hexamer phasing in the promoter region is maintained. Altogether, the findings deepen and refine our insight into structure and length constraints within the EBOV transcription and replication promoter and suggest a remarkable flexibility of the viral polymerase in recognition of PE1 and PE2.

摘要

埃博拉病毒 (EBOV) RNA 有可能在基因组和反基因组/mRNA 水平的转录起始序列 (TSS) 和内部基因的重新启动位点形成发夹结构。建议涉及 TSS 和启动子元件 (PE) 1 和 2 之间的间隔序列的发夹形成调节病毒转录。在这里，我们提供了证据表明这种 RNA 结构在病毒聚合酶的 RNA 合成过程中形成并影响其活性。这是使用在 TSS 间隔区中携带发夹结构变体的单顺反子微型基因组进行分析的，这些变体的长度和稳定性不同。通过报告基因活性和不同病毒 RNA 种类的 qRT-PCR 定量测量转录和复制。我们证明病毒 RNA 合成对高达 ~54 nt 的间隔区延伸具有显着的耐受性，但下降超过此长度限制（66-nt 延伸约 25% 的残留活性）。发现 TSS 间隔区和 mRNA/抗原组水平上发夹结构的微小增量稳定化可迅速消除病毒聚合酶活性，这可用于抑制病毒 RNA 合成的反义策略。最后，当 TSS 处的任何 RNA 结构形成潜力被消除时，病毒转录和复制仍然会发生平衡，前提是启动子区域的六聚体定相得以维持。总而言之，这些发现加深和完善了我们对 EBOV 转录和复制启动子内结构和长度限制的洞察，并表明病毒聚合酶在识别 PE1 和 PE2 方面具有显着的灵活性。