A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport.
A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport.
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DOI：
10.3389/fcell.2022.1011221
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发表时间：
2022
影响因子：
5.5
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中科院分区：
文献类型：
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持续的 SARS-CoV-2/COVID-19 大流行引发了全球公共卫生危机。然而，每个人对 SARS-CoV-2 感染的反应各不相同，不同的病毒变种赋予不同的致病性。因此，必须了解病毒决定因素如何导致 COVID-19。病毒 ORF3a 蛋白是这些病毒决定因素之一，因为其功能与诱导细胞和组织损伤、疾病严重程度和细胞因子风暴有关，而细胞因子风暴是 COVID-19 相关死亡的主要原因。 ORF3a 是一种膜相关蛋白。合成后，它从内质网、高尔基体转运到质膜和亚细胞内膜，包括内体和溶酶体。然而，ORF3a如何在细胞内运输仍然难以捉摸。本研究的目的是进行系统诱变研究，以确定 ORF3a 蛋白与其亚细胞位置的结构关系。在推定的功能相关基序和其他感兴趣区域中产生单氨基酸（aa）和缺失突变。与野生型 ORF3a 相比，使用免疫荧光和 ImageJ 分析来确定和定量 ORF3a 突变体的亚细胞位置。野生型 ORF3a 主要定位在内体和溶酶体的膜上（皮尔逊系数约为 0.8）。与早期的发现一致，蛋白质输出所需的 YXXΦ 基序的删除，在高尔基体中保留了 ORF3a。有趣的是，双甘氨酸（diG）区域（aa 187-188）的突变表现出与 YXXΦ 缺失相似的表型，表明 diG 基序在细胞内转运中具有相似的作用。事实上，中断两个甘氨酸残基中的任何一个，例如删除单个 (dG188)、两个 (dG187/dG188) 或这些残基的替换 (G188Y)，都会导致 ORF3a 保留在高尔基体中（皮尔逊系数≥0.8）。结构分析进一步表明，diG 基序支持反平行 β4 和 β5 片之间的 II 型 β 转角，并通过两个单体之间的氢键连接到 YXXΦ 基序。 diG-YXXΦ 相互作用形成手拉手构型，可以促进二聚化。总之，这些观察结果表明 diG 基序在 ORF3a 细胞内转运中的功能作用。
The ongoing SARS-CoV-2/COVID-19 pandemic caused a global public health crisis. Yet, everyone’s response to SARS-CoV-2 infection varies, and different viral variants confer diverse pathogenicity. Thus, it is imperative to understand how viral determinants contribute to COVID-19. Viral ORF3a protein is one of those viral determinants, as its functions are linked to induction of cell and tissues damages, disease severity and cytokine storm that is a major cause of COVID-19-related death. ORF3a is a membrane-associated protein. Upon synthesis, it is transported from endoplasmic reticulum, Golgi apparatus to plasma membrane and subcellular endomembranes including endosomes and lysosomes. However, how ORF3a is transported intracellularly remains elusive. The goal of this study was to carry out a systematic mutagenesis study to determine the structural relationship of ORF3a protein with its subcellular locations. Single amino acid (aa) and deletion mutations were generated in the putative function-relevant motifs and other regions of interest. Immunofluorescence and ImageJ analyses were used to determine and quantitate subcellular locations of ORF3a mutants in comparison with wildtype ORF3a. The wildtype ORF3a localizes predominantly (Pearson’s coefficients about 0.8) on the membranes of endosomes and lysosomes. Consistent with earlier findings, deletion of the YXXΦ motif, which is required for protein export, retained ORF3a in the Golgi apparatus. Interestingly, mutations in a double glycine (diG) region (aa 187–188) displayed a similar phenotype to the YXXΦ deletion, implicating a similar role of the diG motif in intracellular transport. Indeed, interrupting any one of the two glycine residues such as deletion of a single (dG188), both (dG187/dG188) or substitution (G188Y) of these residues led to ORF3a retention in the Golgi apparatus (Pearson’s coefficients ≥0.8). Structural analyses further suggest that the diG motif supports a type-II β-turn between the anti-parallel β4 and β5 sheets and connects to the YXXΦ motif via hydrogen bonds between two monomers. The diG- YXXΦ interaction forms a hand-in-hand configuration that could facilitate dimerization. Together, these observations suggest a functional role of the diG motif in intracellular transport of ORF3a.