John Sinu P, Wang Tan, Steffen Scott, Longhi Sonia, Schmaljohn Connie S, Jonsson Colleen B
Graduate Program in Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Virol. 2007 Sep;81(17):8967-76. doi: 10.1128/JVI.02523-06. Epub 2007 Jun 13.
The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn(2+), but not Mg(2+), enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.
埃博拉病毒（EBOV）基因组编码几种蛋白质，这些蛋白质对于病毒RNA在体外的复制和转录是必需且足够的；包括核蛋白（NP）、病毒蛋白30（VP30）、病毒蛋白35（VP35）和依赖RNA的RNA聚合酶（L）。VP30通过与第一个转录起始位点上游的RNA二级结构反式作用来调节转录。使用生物信息学方法，我们在VP30的N端鉴定出一个区域，其序列特征典型地代表了内在无序区域和一个假定的RNA结合位点。为了通过实验评估VP30与病毒RNA直接相互作用的能力，我们将重组EBOV VP30纯化至>90%的纯度，并通过紫外线交联和滤膜结合试验评估RNA结合情况。VP30是一种强嗜酸性蛋白；随着pH值降低，RNA结合变得更强。锌离子（Zn²⁺）而非镁离子（Mg²⁺）增强了活性。VP30对转录的增强需要位于前导区域核苷酸54至80内的一个RNA茎环结构。VP30对单独预测的茎环结构或双链RNA显示出低结合亲和力，但当置于上游和下游序列背景中时，对该茎环结构显示出良好的结合亲和力。为了定位与RNA相互作用的区域，我们构建、纯化并检测了一系列VP30的N端缺失突变体与RNA的结合情况。支持RNA结合活性的关键氨基酸定位于26至40位残基，这是一个富含精氨酸的区域。因此，我们首次展示了EBOV VP30与RNA的直接相互作用以及N端区域对结合RNA的重要性。
