| Autor: |
Nelson CR; Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada., Mrozowich T; Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada., Park SM; Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada., D'souza S; Department of Microbiology, Immunology and Infectious Disease, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada., Henrickson A; Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada., Vigar JRJ; Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada., Wieden HJ; Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada., Owens RJ; Research Complex at Harwell, R92 Rutherford Appleton Laboratories, Harwell, Oxford OX1 0QX, UK., Demeler B; Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada.; Department of Chemistry and Biochemistry, University of Montana, Missoula, MT 59812, USA.; NorthWest Biophysics Consortium, University of Lethbridge, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada., Patel TR; 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.; Li Ka Shing Institute of Virology and Discovery Lab, University of Alberta, Edmonton, AB T6G 2E1, Canada. |
| Abstrakt: |
Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae 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 135-555 ) and RVFV non-coding RNAs, IGR and 5' NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17 135-555 , 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 135-555 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 135-555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17 135-555 interacting with and unwinding RVFV non-coding regions. |
