Nelson Corey R, Mrozowich Tyler, Park Sean M, D'souza Simmone, Henrickson Amy, Vigar Justin R J, Wieden Hans-Joachim, Owens Raymond J, Demeler Borries, Patel Trushar R
Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada.
Department of Microbiology, Immunology and Infectious Disease, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada.
Int J Mol Sci. 2020 Dec 23;22(1):54. doi: 10.3390/ijms22010054.
Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5' non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17) and RVFV non-coding RNAs, IGR and 5' NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17, IGR, and 5' NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5' NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17 can unwind both RNAs. Overall, our study provides direct evidence of DDX17 interacting with and unwinding RVFV non-coding regions.
裂谷热病毒（RVFV）是一种由蚊子传播的病毒，属于布尼亚病毒科，可在人类和反刍动物中导致高死亡率和高发病率。先前的研究表明，DEAD盒解旋酶17（DDX17）通过识别基因组S片段中的两个主要非编码RNA来限制RVFV复制：基因间区域（IGR）和5'非编码区域（NCR）。然而，我们缺乏对DDX17与RVFV非编码RNA直接结合的分子见解，以及关于DDX17解开这两种非编码RNA的信息。因此，我们对DDX17解旋酶结构域（DDX17）与RVFV非编码RNA（IGR和5' NCR）进行了广泛的生物物理分析。使用分析超速离心进行的均一性研究表明，DDX17、IGR和5' NCR是纯的。接下来，我们进行了小角X射线散射（SAXS）实验，结果表明DDX17和这两种RNA也是均一的。SAXS分析还表明，DDX17在一定程度上呈球状，而RNA在溶液中呈伸展构象。随后，进行了微量热泳（MST）实验，以研究DDX17与非编码RNA的直接结合。MST实验表明，DDX17与IGR和5' NCR结合，解离常数分别为5.77±0.15 μM和9.85±0.11 μM。由于DDX17是一种RNA解旋酶，我们接下来确定它是否能解开IGR和NCR。我们开发了一种使用MST和荧光标记寡核苷酸的解旋酶检测方法，结果表明DDX17可以解开这两种RNA。总体而言，我们的研究提供了DDX17与RVFV非编码区域相互作用并解开它们的直接证据。
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