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. 2021 Jan 2;22(1):413. doi: 10.3390/ijms22010413.
Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5' 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' TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5' TR of JEV and demonstrated its direct interaction with recombinant DDX3X (K of 1.66 ± 0.21 µM) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5' and 3' TRs of flaviviruses, we investigated if the ZIKV 5' TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5' TR with a K of 7.05 ± 0.75 µM. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5' 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.
黄病毒属包括许多致命病毒，如日本脑炎病毒（JEV）和寨卡病毒（ZIKV）。黄病毒的 5' 端区域（TR）与人类蛋白相互作用，这种相互作用对病毒复制至关重要。已鉴定出与 JEV 的 5' TR 相互作用的人类蛋白之一是 DEAD -box 解旋酶，DDX3X。在这项研究中，我们体外转录了 JEV 的 5' TR，并使用微尺度热泳动（MST）证明了其与重组 DDX3X 的直接相互作用（Kd 为 1.66±0.21 µM）。由于黄病毒的 5' 和 3' TR 具有结构相似性，我们研究了 ZIKV 5' TR 是否也可以与人类 DDX3X 相互作用。我们的 MST 研究表明，DDX3X 识别 ZIKV 5' TR 的 Kd 为 7.05±0.75 µM。接下来，我们进行了解旋酶测定，表明 DDX3X 的结合导致 JEV 和 ZIKV 5' TR 的解旋。总的来说，我们的数据首次表明，DDX3X 可以直接结合并在体外转录的黄病毒 TR 上解旋。总之，我们的工作表明，DDX3X 可以作为抑制黄病毒复制的治疗靶点进一步探索。
