Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong, China.
J Virol. 2022 Apr 27;96(8):e0027922. doi: 10.1128/jvi.00279-22. Epub 2022 Mar 30.
Sp100 (speckled protein 100 kDa) is a constituent component of nuclear structure PML (promyelocytic leukemia) bodies, playing important roles in mediating intrinsic and innate immunity. The Sp100 gene encodes four isoforms with distinct roles in the transcriptional regulation of both cellular and viral genes. Since Sp100 is a primary intranuclear target of infected-cell protein 0 (ICP0), an immediate early E3 ligase encoded by herpes simplex virus 1 (HSV-1), previous investigations attempting to analyze the functions of individual Sp100 variants during HSV-1 infection mostly avoided using a wild-type virus. Therefore, the role of Sp100 under natural infection by HSV-1 remains to be clarified. Here, we reappraised the antiviral capacity of four Sp100 isoforms during infection by a nonmutated HSV-1, examined the molecular behavior of the Sp100 protein in detail, and revealed the following intriguing observations. First, Sp100 isoform A (Sp100A) inhibited wild-type HSV-1 propagation in HEp-2, Sp100, and PML cells. Second, endogenous Sp100 is located in both the nucleus and the cytoplasm. During HSV-1 infection, the nuclear Sp100 level decreased drastically upon the detection of ICP0 in the same subcellular compartment, but cytosolic Sp100 remained stable. Third, transfected Sp100A showed subcellular localizations similar to those of endogenous Sp100 and matched the protein size of endogenous cytosolic Sp100. Fourth, HSV-1 infection induced increased secretion of endogenous Sp100 and ectopically expressed Sp100A, which copurified with extracellular vesicles (EVs) but not infectious virions. Fifth, the Sp100A level in secreting cells positively correlated with its level in EVs, and EV-associated Sp100A restricted HSV-1 in recipient cells. Previous studies show that the PML body component Sp100 protein is immediately targeted by ICP0 of HSV-1 in the nucleus during productive infection. Therefore, extensive studies investigating the interplay of Sp100 isoforms with HSV-1 were conducted using a mutant virus lacking ICP0 or in the absence of infection. The role of Sp100 variants during natural HSV-1 infection remains blurry. Here, we report that Sp100A potently and independently inhibited wild-type HSV-1 and that during HSV-1 infection, cytosolic Sp100 remained stable and was increasingly secreted into the extracellular space, in association with EVs. Furthermore, the Sp100A level in secreting cells positively correlated with its level in EVs and the anti-HSV-1 potency of these EVs in recipient cells. In summary, this study implies an active antiviral role of Sp100A during wild-type HSV-1 infection and reveals a novel mechanism of Sp100A to restrict HSV-1 through extracellular communications.
Sp100（斑点蛋白 100kDa）是核结构 PML（早幼粒细胞白血病）体的组成成分，在介导固有和先天免疫中发挥重要作用。Sp100 基因编码四个具有不同功能的异构体，可调节细胞和病毒基因的转录调控。由于 Sp100 是单纯疱疹病毒 1（HSV-1）感染细胞蛋白 0（ICP0）的主要核内靶标，是 ICP0 编码的一种早期 E3 连接酶，因此，之前试图在 HSV-1 感染期间分析单个 Sp100 变体功能的研究大多避免使用野生型病毒。因此，Sp100 在 HSV-1 自然感染下的作用仍有待阐明。在这里，我们重新评估了非突变 HSV-1 感染期间四个 Sp100 异构体的抗病毒能力，详细研究了 Sp100 蛋白的分子行为，并揭示了以下有趣的观察结果。首先，Sp100 异构体 A（Sp100A）抑制 HEp-2、Sp100 和 PML 细胞中的野生型 HSV-1 增殖。其次，内源性 Sp100 存在于核内和细胞质中。在 HSV-1 感染期间，当同一亚细胞隔室中检测到 ICP0 时，核内 Sp100 水平急剧下降，但细胞质 Sp100 保持稳定。第三，转染的 Sp100A 显示出与内源性 Sp100 相似的亚细胞定位，并与内源性细胞质 Sp100 的蛋白大小相匹配。第四，HSV-1 感染诱导内源性 Sp100 和异位表达的 Sp100A 的大量分泌，这些蛋白与细胞外囊泡（EVs）而不是感染性病毒粒子共纯化。第五，分泌细胞中的 Sp100A 水平与其在 EVs 中的水平呈正相关，EV 相关的 Sp100A 限制了受感染细胞中的 HSV-1。以前的研究表明，在产毒感染期间，HSV-1 的 PML 体成分 Sp100 蛋白立即被 ICP0 靶向细胞核。因此，广泛的研究使用缺乏 ICP0 的突变病毒或在没有感染的情况下研究 Sp100 异构体与 HSV-1 的相互作用。Sp100 变体在自然 HSV-1 感染期间的作用仍然模糊不清。在这里，我们报告 Sp100A 强烈且独立地抑制野生型 HSV-1，并且在 HSV-1 感染期间，细胞质 Sp100 保持稳定，并与 EV 一起越来越多地分泌到细胞外空间。此外，分泌细胞中 Sp100A 的水平与其在 EVs 中的水平呈正相关，并且这些 EVs 在受感染细胞中的抗 HSV-1 效力。总之，这项研究表明 Sp100A 在野生型 HSV-1 感染期间发挥积极的抗病毒作用，并揭示了 Sp100A 通过细胞外通讯限制 HSV-1 的新机制。
