Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, and Shanghai Institute of Infectious Disease and Biosecurity, Fudan University, Shanghai, China.
Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.
J Virol. 2024 May 14;98(5):e0034924. doi: 10.1128/jvi.00349-24. Epub 2024 Apr 19.
The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel coronavirus severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has rapidly spread worldwide since its emergence in late 2019. Its ongoing evolution poses challenges for antiviral drug development. Coronavirus nsp6, a multiple-spanning transmembrane protein, participates in the biogenesis of the viral replication complex, which accommodates the viral replication-transcription complex. The roles of its structural domains in viral replication are not well studied. Herein, we predicted the structure of the SARS-CoV-2 nsp6 protein using AlphaFold2 and identified a highly folded C-terminal region (nsp6C) downstream of the transmembrane helices. The enhanced green fluorescent protein (EGFP)-fused nsp6C was found to cluster in the cytoplasm and associate with membranes. Functional mapping identified a minimal membrane-associated element (MAE) as the region from amino acids 237 to 276 (LGV-KLL), which is mainly composed of the α-helix H1 and the α-helix H2; the latter exhibits characteristics of an amphipathic helix (AH). Mutagenesis studies and membrane flotation experiments demonstrate that AH-like H2 is required for MAE-mediated membrane association. This MAE was functionally conserved across MERS-CoV, HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63, all capable of mediating membrane association. In a SARS-CoV-2 replicon system, mutagenesis studies of H2 and replacements of H1 and H2 with their homologous counterparts demonstrated requirements of residues on both sides of the H2 and properly paired H1-H2 for MAE-mediated membrane association and viral replication. Notably, mutations I266A and K274A significantly attenuated viral replication without dramatically affecting membrane association, suggesting a dual role of the MAE in viral replication: mediating membrane association as well as participating in protein-protein interactions.IMPORTANCESevere acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) assembles a double-membrane vesicle (DMV) by the viral non-structural proteins for viral replication. Understanding the mechanisms of the DMV assembly is of paramount importance for antiviral development. Nsp6, a multiple-spanning transmembrane protein, plays an important role in the DMV biogenesis. Herein, we predicted the nsp6 structure of SARS-CoV-2 and other human coronaviruses using AlphaFold2 and identified a putative membrane-associated element (MAE) in the highly conserved C-terminal regions of nsp6. Experimentally, we verified a functionally conserved minimal MAE composed of two α-helices, the H1, and the amphipathic helix-like H2. Mutagenesis studies confirmed the requirement of H2 for MAE-mediated membrane association and viral replication and demonstrated a dual role of the MAE in viral replication, by mediating membrane association and participating in residue-specific interactions. This functionally conserved MAE may serve as a novel anti-viral target.
新型冠状病毒病 2019（COVID-19）疫情由新型冠状病毒严重急性呼吸系统综合征冠状病毒 2 型（SARS-CoV-2）引起，自 2019 年底出现以来已迅速在全球蔓延。其持续进化给抗病毒药物的开发带来了挑战。冠状病毒 nsp6 是一种多跨膜蛋白，参与病毒复制复合物的生物发生，该复合物容纳病毒复制-转录复合物。其结构域在病毒复制中的作用尚未得到很好的研究。在此，我们使用 AlphaFold2 预测了 SARS-CoV-2 nsp6 蛋白的结构，并鉴定出跨膜螺旋下游具有高度折叠的 C 末端区域（nsp6C）。发现融合增强型绿色荧光蛋白（EGFP）的 nsp6C 在细胞质中聚集并与膜结合。功能映射确定了一个最小的膜相关元件（MAE）作为氨基酸 237 到 276（LGV-KLL）的区域，该区域主要由α-螺旋 H1 和α-螺旋 H2 组成；后者表现出两性螺旋（AH）的特征。突变和膜浮选实验表明，AH 样 H2 是 MAE 介导的膜结合所必需的。该 MAE 在 MERS-CoV、HCoV-OC43、HCoV-229E、HCoV-HKU1 和 HCoV-NL63 中均具有功能保守性，这些病毒均能够介导膜结合。在 SARS-CoV-2 复制子系统中，对 H2 的突变研究以及用同源物替代 H1 和 H2，表明 H2 两侧的残基以及正确配对的 H1-H2 对于 MAE 介导的膜结合和病毒复制都是必需的。值得注意的是，I266A 和 K274A 突变显著削弱了病毒复制，而对膜结合没有明显影响，这表明 MAE 在病毒复制中具有双重作用：介导膜结合以及参与蛋白质-蛋白质相互作用。重要性：严重急性呼吸系统综合征冠状病毒 2 型（SARS-CoV-2）通过病毒非结构蛋白组装双膜囊泡（DMV）进行病毒复制。了解 DMV 组装的机制对于抗病毒药物的开发至关重要。nsp6 是一种多跨膜蛋白，在 DMV 生物发生中发挥重要作用。在此，我们使用 AlphaFold2 预测了 SARS-CoV-2 和其他人类冠状病毒的 nsp6 结构，并在 nsp6 的高度保守 C 末端区域鉴定出一个假定的膜相关元件（MAE）。实验验证了一个由两个α-螺旋，H1 和类两性螺旋的 H2 组成的具有功能保守性的最小 MAE。突变研究证实了 H2 对于 MAE 介导的膜结合和病毒复制的必要性，并证明了 MAE 在病毒复制中的双重作用，通过介导膜结合和参与特定残基的相互作用。这个具有功能保守性的 MAE 可能成为一种新型的抗病毒靶点。
