Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

登革病毒（Dengue virus, DENV）是引发人类致死性疾病的最重要的节肢动物传播病原体之一。目前尚无针对登革热的疫苗或特异性抗病毒治疗药物。与其他RNA病毒类似，固有免疫系统在调控登革病毒感染及疾病转归过程中发挥关键作用。尽管作为宿主保护性免疫核心的干扰素（Interferon, IFN）应答已被证实可限制登革病毒复制，但干扰素处理调控登革病毒感染的分子机制细节仍有待阐明。
本研究通过采用经I型干扰素处理的细胞来源cDNA文库开展功能获得性筛选，鉴定出一个此前未被表征的基因C19orf66，其属于可抑制登革病毒复制的干扰素刺激基因（IFN-stimulated gene, ISG），我们将其命名为登革病毒产量抑制因子（Repressor of yield of DENV, RyDEN）。
过表达与基因敲低实验结果显示，RyDEN的表达可使人源细胞对所有血清型登革病毒产生抗性。此外，RyDEN的表达还可限制丙型肝炎病毒、昆津病毒、基孔肯雅病毒、1型单纯疱疹病毒及人腺病毒的复制。
尤为关键的是，RyDEN被认定为干扰素介导的抗登革病毒应答中的核心效应分子。通过亲和纯化-质谱分析，研究发现RyDEN可与细胞mRNA结合蛋白、聚(A)结合蛋白胞质1（poly(A)-binding protein cytoplasmic 1, PABPC1）以及La基序相关蛋白1（La motif-related protein 1, LARP1）形成复合物。有趣的是，PABPC1与LARP1被证实为登革病毒复制的正向调控因子。
鉴于RyDEN可影响登革病毒复制的胞内事件，且在表达RyDEN的细胞中亦可观察到基于登革病毒报告载体RNA的蛋白质合成受到抑制，本研究结果提示，RyDEN或可通过与病毒RNA及细胞mRNA结合蛋白相互作用，干扰登革病毒的翻译过程，进而抑制感染细胞内的病毒复制。