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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&prime, 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 (DDX17135&ndash, 555) and RVFV non-coding RNAs, IGR and 5&rsquo, NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135&ndash, 555, IGR, and 5&rsquo, 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&rsquo, NCR with a dissociation constant of 5.77 &plusmn, 0.15 &micro, M and 9.85 &plusmn, 0.11 &micro, M, respectively. As DDX17135&ndash, 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 DDX17135&ndash, 555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135&ndash, 555 interacting with and unwinding RVFV non-coding regions.

Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5&prime, terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5&prime, TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5&prime, TR of JEV and demonstrated its direct interaction with recombinant DDX3X (Kd of 1.66 &plusmn, 0.21 &micro, M) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5&prime, and 3&prime, TRs of flaviviruses, we investigated if the ZIKV 5&prime, TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5&prime, TR with a Kd of 7.05 &plusmn, 0.75 &micro, M. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5&prime, TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication