Endoplasmic reticulum-associated SARS-CoV-2 ORF3a elicits heightened cytopathic effects despite robust ER-associated degradation.
Endoplasmic reticulum-associated SARS-CoV-2 ORF3a elicits heightened cytopathic effects despite robust ER-associated degradation.
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严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) ORF3a 蛋白在病毒发病机制和 2019 年冠状病毒病 (COVID-19) 中发挥着至关重要的作用。与刺突蛋白一样，ORF3a 经常发生突变，并且某些变异与 COVID-19 的严重程度有关。鉴于ORF3a突变的临床意义和功能意义，我们针对各种已知的功能元件进行了全面的诱变研究，并根据其亚细胞定位揭示了两种独特类型的ORF3a蛋白：ORF3a蛋白主要定位于溶酶体膜（L-ORF3a）和存在于内质网中的蛋白（E-ORF3a）。本研究的目的是对比这两种类型的 ORF3a 蛋白之间的功能和机制差异。我们检查了六种不同的 ORF3a 突变体，并评估了它们对细胞氧化应激、核因子 kappa B 诱导的细胞因子产生和细胞死亡的影响。从机制上讲，我们探索了 ORF3a 诱导的内质网应激、自噬以及与相关细胞蛋白的相互作用。我们的研究结果表明，ORF3a 蛋白通过类似的机制诱导细胞病变效应，无论其亚细胞位置如何。然而，与 L-ORF3a 蛋白相比，E-ORF3a 蛋白尽管丰度较低且对 ER 应激和自噬的影响最小，但仍会引起更明显的细胞病变效应。这种差异归因于 ER 相关降解，因为 ORF3a 蛋白与泛素 E3 连接酶 TRIM59 结合。 26S 蛋白酶体的抑制可部分恢复 E-ORF3a 的蛋白质水平和细胞内质网应激反应。这表明，由于 ER 的脆弱性质，即使少量的 ORF3a 也可能导致显着的细胞病变效应。我们的研究强调了这两个亚细胞区室中动态细胞信号传导响应 ORF3a 的复杂相互作用。严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 大流行已通过 2019 冠状病毒病 (COVID-19) 不幸夺去了数百万人的生命，而我们对导致相关死亡的精确分子机制的理解仍然存在重大差距。人们感兴趣的一个关键病毒因子是 SARS-CoV-2 ORF3a 蛋白，它已被确定为宿主细胞促炎症反应的有效诱导剂，能够引发灾难性的细胞因子风暴，这是导致与 COVID-19 相关的死亡的主要原因。此外，ORF3a 与刺突蛋白非常相似，表现出频繁突变的倾向，其中某些变异与 COVID-19 的严重程度有关。我们之前的研究揭示了两种不同类型的 ORF3a 突变蛋白，按其亚细胞定位进行分类，为对这两种类型的 ORF3a 变体之间的功能和机制差异进行比较研究奠定了基础。鉴于天然 ORF3a 突变的临床意义和功能意义，这项研究的结果有望为了解这些突变 ORF3a 蛋白在 COVID-19 发病机制中的潜在作用提供宝贵的见解。
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF3a protein plays a vital role in viral pathogenesis and coronavirus disease 2019 (COVID-19). Like the spike protein, ORF3a mutates frequently, and certain variants are associated with the severity of COVID-19. Given the clinical significance and functional implications of ORF3a mutations, we conducted a comprehensive mutagenesis study targeting various known functional elements and revealed two distinctive types of ORF3a proteins based on their subcellular localizations: ORF3a proteins primarily localize on the lysosomal membrane (L-ORF3a) and those present in the endoplasmic reticulum (E-ORF3a). The objective of this study was to contrast the functional and mechanistic distinctions between these two types of ORF3a proteins. We examined six distinct ORF3a mutants and assessed their effects on cellular oxidative stress, nuclear factor kappa B-induced cytokine production, and cell death. Mechanistically, we explored ORF3a-induced ER stress, autophagy, and interactions with relevant cellular proteins. Our findings indicate that ORF3a proteins induce cytopathic effects through a similar mechanism, irrespective of their subcellular location. However, E-ORF3a proteins elicit more pronounced cytopathic effects despite their lower abundance and minimal impact on ER stress and autophagy when compared to L-ORF3a proteins. This discrepancy is attributed to ER-associated degradation since ORF3a proteins bind to a ubiquitin E3 ligase TRIM59. Inhibition of the 26S proteasome partially restores the protein levels of E-ORF3a and cellular ER stress response. This suggests that even a small quantity of ORF3a can lead to significant cytopathic effects due to the delicate nature of ER. Our study underscores the intricate interplay of dynamic cellular signaling within these two subcellular compartments in response to ORF3a. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has tragically claimed millions of lives through coronavirus disease 2019 (COVID-19), and there remains a critical gap in our understanding of the precise molecular mechanisms responsible for the associated fatality. One key viral factor of interest is the SARS-CoV-2 ORF3a protein, which has been identified as a potent inducer of host cellular proinflammatory responses capable of triggering the catastrophic cytokine storm, a primary contributor to COVID-19-related deaths. Moreover, ORF3a, much like the spike protein, exhibits a propensity for frequent mutations, with certain variants linked to the severity of COVID-19. Our previous research unveiled two distinct types of ORF3a mutant proteins, categorized by their subcellular localizations, setting the stage for a comparative investigation into the functional and mechanistic disparities between these two types of ORF3a variants. Given the clinical significance and functional implications of the natural ORF3a mutations, the findings of this study promise to provide invaluable insights into the potential roles undertaken by these mutant ORF3a proteins in the pathogenesis of COVID-19.
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