2019年冠状病毒病(COVID-19)大流行,由新型冠状病毒引起的严重急性呼吸综合征冠状病毒2型(SARS-CoV-2),自2019年底出现以来,它在全球范围内迅速传播。它的持续发展对抗病毒药物的开发提出了挑战。冠状病毒nsp6,一种跨膜蛋白,参与病毒复制复合体的生物发生,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。在这里,我们使用AlphaFold2预测了SARS-CoV-2nsp6蛋白的结构,并鉴定了跨膜螺旋下游的高度折叠的C末端区域(nsp6C)。发现增强的绿色荧光蛋白(EGFP)融合的nsp6C在细胞质中聚集并与膜结合。功能作图确定了一个最小的膜相关元件(MAE)作为氨基酸237至276(LGV-KLL)的区域,主要由α-螺旋H1和α-螺旋H2组成;后者表现出两亲性螺旋(AH)的特征。诱变研究和膜浮选实验表明,MAE介导的膜缔合需要AH样H2。该MAE在MERS-CoV中功能保守,HCoV-OC43,HCoV-229E,HCoV-HKU1和HCoV-NL63均能够介导膜缔合。在SARS-CoV-2复制子系统中,H2的诱变研究以及H1和H2与同源对应物的置换表明,H2两侧的残基都需要,并且H1-H2正确配对以进行MAE介导的膜缔合和病毒复制。值得注意的是,突变I266A和K274A显着减弱病毒复制,而不会显着影响膜关联,提示MAE在病毒复制中的双重作用:介导膜缔合以及参与蛋白质-蛋白质相互作用。重要急性呼吸综合征冠状病毒2型(SARS-CoV-2)通过病毒非结构蛋白组装双膜囊泡(DMV)用于病毒复制。了解DMV组装的机制对于抗病毒开发至关重要。Nsp6,一种跨膜蛋白,在DMV的生物发生中起着重要作用。在这里,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的高度保守的C端区域中鉴定了推定的膜相关元件(MAE)。实验上,我们验证了由两个α-螺旋组成的功能保守的最小MAE,H1和两亲性螺旋状H2。诱变研究证实了MAE介导的膜缔合和病毒复制需要H2,并证明了MAE在病毒复制中的双重作用。通过介导膜缔合和参与残基特异性相互作用。这种功能保守的MAE可以作为新的抗病毒靶标。
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.