Feinberg Mandi A, Le My T, Carpio Kassandra L, Knyazhanskaya Ekaterina, Barrett Alan D T, Choi Kyung H
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.
Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas, USA.
J Virol. 2025 Aug 18:e0202324. doi: 10.1128/jvi.02023-24.
West Nile virus (WNV) is a single-stranded, positive-sense RNA virus in the genus, within . The genus encompasses numerous pathogens of public health importance, including WNV, dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV). Orthoflavivirus replication depends on the presence of the stem-loop A (SLA) structure in the 5' untranslated region of the genome. The viral polymerase, NS5, interacts with the SLA and initiates synthesis of the negative-strand RNA. The sequences and secondary structures of SLA and NS5 are highly conserved across orthoflaviviruses, suggesting that the viruses utilize a similar SLA-mediated replication mechanism. Here, we determined the molecular shapes of WNV and JEV SLAs and investigated WNV NS5 interaction with orthoflavivirus SLAs. Although WNV NS5 interacts with DENV, ZIKV, and JEV SLAs in binding assays, only DENV and ZIKV SLAs could replace WNV SLA for viral replication. Next, we found that the top and side loops of SLA are important regions for WNV NS5 interaction. Consequently, when these SLA mutations were introduced into a WNV replicon, genomic replication was greatly reduced. Finally, we tested whether the WNV SLA mimic could inhibit viral replication. The addition of exogenous SLA reduces replication of both WNV replicon and infectious virus, suggesting that exogenous SLA can outcompete the viral SLA for NS5 interaction. Next-generation sequencing data indicate that the presence of exogenous SLA during infection increased the genetic diversity of WNV.IMPORTANCEWest Nile virus (WNV) causes West Nile disease in humans. Approximately 1 in 150 cases develops serious neurological complications, such as meningitis or encephalitis. Currently, no vaccines or antiviral treatments are available. WNV relies on a conserved RNA element in the genome, known as stem-loop A (SLA), to recruit viral polymerase for replication. We found that WNV polymerase can bind the SLAs of other orthoflaviviruses, including dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV). However, only the DENV and ZIKV SLAs supported replication when substituted into a WNV replicon. The failure of the JEV SLA to support WNV replication suggests that efficient replication requires additional virus-specific factors beyond the polymerase-SLA interaction. We then tested whether exogenous SLA could act as an RNA decoy to compete with genomic SLA and inhibit viral replication. The addition of SLA RNA in virus-infected cells significantly reduced viral replication and infection, highlighting the therapeutic potential of viral RNA mimic against WNV.
西尼罗河病毒（WNV）是黄病毒属中的一种单链、正义RNA病毒。该属包含许多对公共卫生具有重要意义的病原体，包括西尼罗河病毒、登革病毒（DENV）、寨卡病毒（ZIKV）和日本脑炎病毒（JEV）。正黄病毒的复制依赖于基因组5'非翻译区中茎环A（SLA）结构的存在。病毒聚合酶NS5与SLA相互作用并启动负链RNA的合成。SLA和NS5的序列及二级结构在正黄病毒中高度保守，这表明这些病毒利用相似的SLA介导的复制机制。在此，我们确定了西尼罗河病毒和日本脑炎病毒SLA的分子形状，并研究了西尼罗河病毒NS5与正黄病毒SLA的相互作用。尽管在结合试验中西尼罗河病毒NS5与登革病毒、寨卡病毒和日本脑炎病毒的SLA相互作用，但只有登革病毒和寨卡病毒的SLA能够替代西尼罗河病毒SLA进行病毒复制。接下来，我们发现SLA的顶部和侧面环是西尼罗河病毒NS5相互作用的重要区域。因此，当将这些SLA突变引入西尼罗河病毒复制子时，基因组复制大幅减少。最后，我们测试了西尼罗河病毒SLA模拟物是否能抑制病毒复制。添加外源性SLA可减少西尼罗河病毒复制子和感染性病毒的复制，这表明外源性SLA可以与病毒SLA竞争NS5相互作用。下一代测序数据表明，感染期间外源性SLA的存在增加了西尼罗河病毒的遗传多样性。
重要性
西尼罗河病毒可导致人类患西尼罗河病。约每150例病例中就有1例会出现严重的神经并发症，如脑膜炎或脑炎。目前，尚无可用的疫苗或抗病毒治疗方法。西尼罗河病毒依赖基因组中一种保守的RNA元件，即茎环A（SLA），来招募病毒聚合酶进行复制。我们发现西尼罗河病毒聚合酶可以结合其他正黄病毒（包括登革病毒、寨卡病毒和日本脑炎病毒）的SLA。然而，只有登革病毒和寨卡病毒的SLA在替代到西尼罗河病毒复制子时能支持复制。日本脑炎病毒SLA无法支持西尼罗河病毒复制，这表明高效复制除了聚合酶 - SLA相互作用外还需要其他病毒特异性因子。然后，我们测试了外源性SLA是否可以作为RNA诱饵与基因组SLA竞争并抑制病毒复制。在病毒感染的细胞中添加SLA RNA可显著降低病毒复制和感染，这突出了病毒RNA模拟物针对西尼罗河病毒的治疗潜力。
