Introduction

Hand, foot, and mouth disease (HFMD) is a common enterovirus-induced illness primarily affecting children under 5 years of age. Enterovirus 71 (EV71) is a major causative agent associated with severe HFMD that can lead to fatal neurological complications. Despite available vaccines, no antiviral therapy with a clearly defined molecular mechanism has received approval. While most antiviral agents target viral components, host-targeting antiviral approaches remain underexplored. We first identified voxtalisib, a dual PI3K/mTOR inhibitor, through compound screening as a potential inhibitor of EV71 replication; however, the underlying mechanism remains unclear.

Objectives

To identify novel host-targeted antiviral candidates and to investigate the antiviral mechanism of voxtalisib against EV71 and other enteroviruses.

Methods

Voxtalisib was first identified as a potent anti-EV71 compound through in vitro phenotypic screening. Follow-up experiments in rhabdomyosarcoma (RD) cells were used to assess the effect of voxtalisib on EV71 replication, and proteomic analysis identified its molecular targets. Voxtalisib’s antiviral activity was also assessed against Coxsackie B viruses (CVB3, CVB4-5, and CVB4-7) and Echovirus 11 (Echo11). In vivo, suckling ICR mice were treated with voxtalisib. Survival rates, viral loads, and histopathological changes were subsequently evaluated.

Results

Proteomics analysis identified Ras-related nuclear protein (RAN) as a key host factor in EV71 replication. EV71 infection significantly upregulated RAN, while voxtalisib suppressed RAN. Mechanistically, RAN regulates nuclear–cytoplasmic transport of phosphorylated STAT1/2 (p-STAT1/2), affecting the interferon (IFN)-mediated antiviral response. Consequently, downregulating RAN expression enhanced the nuclear retention of p-STAT1/2 and upregulated interferon-stimulated genes expression, ultimately reducing EV71 replication. In vivo, voxtalisib improved survival, decreased viral loads, and alleviated organ damage in EV71-infected ICR suckling mice. Similar RAN-dependent p-STAT nuclear retention and antiviral effects were also observed against CVB and Echo11, confirming voxtalisib’s broad-spectrum antiviral potential.

Conclusion

RAN is a novel antiviral host target that indirectly mediates EV71 replication by regulating the nuclear–cytoplasmic transport of p-STAT1/2. Voxtalisib effectively restores IFN–STAT signaling by modulating RAN, offering a promising host-directed antiviral strategy against enteroviruses.

中文翻译：

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Voxtalisib 通过下调宿主 RAN 和恢复 IFN-STAT 信号传导来抑制肠道病毒 71 复制

  介绍

手足口病 （HFMD） 是一种常见的肠道病毒诱发的疾病，主要影响 5 岁以下儿童。肠道病毒 71 （EV71） 是与严重手足口病相关的主要病原体，可导致致命的神经系统并发症。尽管有可用的疫苗，但没有具有明确分子机制的抗病毒疗法获得批准。虽然大多数抗病毒药物靶向病毒成分，但靶向宿主的抗病毒方法仍未得到充分探索。我们首先通过化合物筛选确定了 voxtalisib，一种双重 PI3K/mTOR 抑制剂，作为 EV71 复制的潜在抑制剂;然而，其潜在机制仍不清楚。

  目标

确定新的宿主靶向抗病毒候选药物，并研究 voxtalisib 对 EV71 和其他肠道病毒的抗病毒机制。

  方法

Voxtalisib 最初通过体外表型筛选被鉴定为一种有效的抗 EV71 化合物。横纹肌肉瘤 （RD） 细胞的随访实验用于评估 voxtalisib 对 EV71 复制的影响，蛋白质组学分析确定了其分子靶点。还评估了 Voxtalisib 对柯萨奇 B 病毒 （CVB3、CVB4-5 和 CVB4-7） 和埃可病毒 11 （Echo11） 的抗病毒活性。 在体内， 用 voxtalisib 处理哺乳 ICR 小鼠。随后评估了存活率、病毒载量和组织病理学变化。

  结果

蛋白质组学分析确定 Ras 相关核蛋白 （RAN） 是 EV71 复制的关键宿主因子。EV71 感染显着上调 RAN，而 voxtalisib 抑制 RAN。从机制上讲，RAN 调节磷酸化 STAT1/2 （p-STAT1/2） 的核质转运，影响干扰素 （IFN） 介导的抗病毒反应。因此，下调 RAN 表达增强了 p-STAT1/2 的核保留，并上调了干扰素刺激的基因表达，最终减少了 EV71 复制。 在体内 ，voxtalisib 提高了 EV71 感染的 ICR 乳鼠的存活率，降低了病毒载量，并减轻了器官损伤。还观察到类似的 RAN 依赖性 p-STAT 核保留和抗病毒作用，针对 CVB 和 Echo11，证实了 voxtalisib 的广谱抗病毒潜力。

  结论

RAN 是一种新型抗病毒宿主靶标，通过调节 p-STAT1/2 的核质转运间接介导 EV71 复制。Voxtalisib 通过调节 RAN 有效恢复 IFN-STAT 信号传导，提供了一种有前途的针对肠道病毒的宿主导向抗病毒策略。