Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8069, St. Louis, MO, 63110, USA.
ICCE Institute, Washington University School of Medicine, Saint Louis, MO, USA.
Sci Rep. 2024 Oct 9;14(1):23533. doi: 10.1038/s41598-024-74477-3.
Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.
病毒感染的识别通常依赖于双链 RNA（dsRNA）的检测，这一过程在许多不同的生物体中都被保守。在哺乳动物中，MDA5、RIG-I、OAS 和 PKR 等蛋白可以检测病毒 dsRNA，但难以区分病毒和内源性 dsRNA。本研究探讨了靶向 DDX54 的 shRNA 激活 PKR 的潜力，PKR 是 dsRNA 免疫反应中的关键分子。特异性 shRNA 敲低 DDX54 可在人细胞中诱导强烈的 PKR 激活，即使在 DDX54 过表达的情况下，提示存在非靶向机制。ADAR1 的敲低增强了 shRNA 对 PKR 的激活，ADAR1 是一种抑制 PKR 激活的 dsRNA 结合蛋白，表明这是一种 dsRNA 介导的机制。体外实验证实了 shRNA 对 PKR 的直接激活。这些发现强调了需要严格的对照和替代方法来验证基因功能，并最小化意外的免疫途径激活。
