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Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the <i>Bunyaviridae</i> 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<sub>135-555</sub>) and RVFV non-coding RNAs, IGR and 5' NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17<sub>135-555</sub>, 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<sub>135-555</sub> 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<sub>135-555</sub> can unwind both RNAs. Overall, our study provides direct evidence of DDX17<sub>135-555</sub> interacting with and unwinding RVFV non-coding regions.

Original publication

DOI

10.3390/ijms22010054

Type

Journal article

Journal

International journal of molecular sciences

Publication Date

23/12/2020

Volume

22

Addresses

Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada.