微生物学报

冠状病毒刺突蛋白亚基2介导病毒膜融合的作用机制及抑制剂研究进展

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李溢璇 , 谢莎 , 何航 , 孙彦刚

微生物学报 | 综述 2026,66(7): 3162-3179

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微生物学报 |综述 2026 , 66 (7) : 3162 -3179

冠状病毒刺突蛋白亚基2介导病毒膜融合的作用机制及抑制剂研究进展

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李溢璇, 谢莎, 何航, 孙彦刚

作者信息

河南中医药大学医学院，河南郑州

作者简介:

作者贡献声明

李溢璇：数据收集，撰写文章；谢莎：格式核对，文章审阅；何航：文章审阅；孙彦刚：提出概念，获取基金，文章审阅。

Research progress in the mechanism of coronavirus spike protein subunit 2 in mediating viral membrane fusion and its inhibitors

Yixuan LI, Sha XIE, Hang HE, Yangang SUN

Affiliations

School of Medicine, Henan University of Chinese Medicine, Zhengzhou, Henan, China

出版时间: 2026-07-04 doi: 10.13343/j.cnki.wsxb.20250828

文章导航

摘要

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冠状病毒感染严重威胁人类生命健康，且对畜牧业造成巨大的经济损失。冠状病毒主要通过细胞表面膜融合途径和内体膜融合途径侵入宿主细胞。在病毒入侵过程中，刺突蛋白亚基1 (spike protein subunit 1, S1)负责识别并结合宿主细胞受体，刺突蛋白亚基2 (spike protein subunit 2, S2)介导病毒膜与宿主细胞膜的融合。由于S2在不同冠状病毒中高度保守，阻断其介导的膜融合过程可有效抑制病毒感染，因此成为广谱抗冠状病毒药物研发的重要靶点。本文综述了近年来冠状病毒入侵宿主细胞的分子机制、S蛋白的结构与功能特征以及膜融合抑制剂的研究进展，并分析了靶向S2药物研发面临的挑战与未来发展方向，以期为冠状病毒的防控策略及新型抗病毒药物研发提供理论参考。

关键词

冠状病毒 / 刺突蛋白 / 刺突蛋白亚基2 / 膜融合 / 抑制剂

Abstract

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Coronavirus infections pose a serious threat to human health and have resulted in substantial economic losses to the livestock industry. Coronaviruses invade host cells primarily through two pathways: cell surface membrane fusion and endosomal membrane fusion. During viral entry, the spike protein subunit 1 (S1) recognizes and binds to cellular receptors, while the spike protein subunit 2 (S2) facilitates membrane fusion between the viral envelope and host cell membrane. Due to its high sequence conservation across different coronaviruses, S2 represents an attractive target for the development of broad-spectrum antiviral agents. Blocking S2-mediated membrane fusion can effectively inhibit viral infection. This review summarizes recent advances in understanding the mechanisms of coronavirus entry into host cells, the structure and function of the spike protein, and the development of membrane fusion inhibitors. In addition, this paper discusses the challenges and future prospects in targeting S2 for antiviral drug development, aiming to provide insights for coronavirus prevention and the discovery of novel antiviral therapeutics.

Key words

coronavirus / spike protein / spike protein subunit 2 / membrane fusion / inhibitor

引用本文

李溢璇, 谢莎, 何航, 孙彦刚. 冠状病毒刺突蛋白亚基2介导病毒膜融合的作用机制及抑制剂研究进展. 微生物学报, 2026 , 66 (7) : 3162 -3179 . DOI: 10.13343/j.cnki.wsxb.20250828

Yixuan LI, Sha XIE, Hang HE, Yangang SUN. Research progress in the mechanism of coronavirus spike protein subunit 2 in mediating viral membrane fusion and its inhibitors[J]. Acta Microbiologica Sinica, 2026 , 66 (7) : 3162 -3179 . DOI: 10.13343/j.cnki.wsxb.20250828

正文

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冠状病毒(coronavirus, CoV)是一类具有广泛宿主范围和传播能力的RNA病毒，其感染可致使禽类和哺乳动物出现严重的呼吸系统、消化系统及神经系统疾病。感染人类的冠状病毒主要有7种，人冠状病毒(human coronavirus, HCoV)-229E、HCoV-NL63、HCoV-OC43和HCoV-HKU1可引发人体轻微上呼吸道疾病；严重急性呼吸综合征冠状病毒(severe acute respiratory syndrome coronavirus, SARS-CoV)、中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)和严重急性呼吸综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)则会导致人体出现严重呼吸道症状和炎症因子风暴，甚至造成患者死亡，给人类生命安全带来巨大威胁^[1-2]。此外，2017-2018年在马来西亚砂拉越邦住院肺炎患者样本中发现了一种新型犬-猫重组α冠状病毒(基因Ⅱ型) CCoV-HuPn-2018，这是首个被证实可感染人类的犬冠状病毒^[3]。Lednicky等^[4]也报道在海地检测到猪德尔塔冠状病毒(porcine delta coronavirus, PDCoV)感染儿童的病例。除感染人类外，冠状病毒对畜牧业危害也极为严重。例如，PDCoV^[5-6]、猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)^[7]和猪肠道α冠状病毒(porcine enteric alphacoronavirus, PEAV) [又称猪急性腹泻综合征冠状病毒(swine acute diarrhea syndrome coronavirus, SADS-CoV)]^[8-9]等可引发仔猪胃肠道疾病，导致动物死亡，给养殖业造成巨大经济损失。当前新出现的SARS-CoV-2 BA.3.2变异株具有很强的免疫逃逸能力^[10]。虽然已研发或筛选出瑞德西韦(Remdesivir)^[11]、Paxlovid^[12]等药物和多款疫苗，但冠状病毒的高变异性仍给疫情防控带来巨大挑战。

冠状病毒是一类具有包膜的正链RNA病毒，隶属于套式病毒目(Nidovirales)、冠状病毒科(Coronaviridae)、正冠状病毒亚科(Orthocoronavirinae)、冠状病毒属(Coronavirus)。依据国际病毒分类委员会的分类标准，冠状病毒科被划分为4个属：α冠状病毒属(alpha-coronavirus, α-CoV)、β冠状病毒属(beta-coronavirus, β-CoV)、γ冠状病毒属(gamma-coronavirus, γ-CoV)和δ冠状病毒属(delta-coronavirus, δ-CoV)^[13]。冠状病毒基因组编码多种蛋白，包括结构蛋白和非结构蛋白，结构蛋白有刺突蛋白(spike protein, S)、包膜蛋白(envelope protein, E)、膜蛋白(membrane protein, M)、核衣壳蛋白(nucleocapsid protein, N)等，HCoV-OC43、HCoV-HKU1和牛冠状病毒(bovine coronavirus, BCoV)等β冠状病毒lineage A (Embecovirus亚属)病毒还表达血凝素酯酶蛋白(hemagglutinin-esterase protein, HE)^[14]。非结构蛋白有RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase, RdRp)、主蛋白酶(Mpro/3CLPro)、木瓜样蛋白酶(papain-like protease, PLpro)等，结构蛋白和非结构蛋白共同参与病毒复制、转录以及组装。

S蛋白是冠状病毒编码的关键结构蛋白之一，可介导病毒感染宿主细胞。S蛋白可分为亚基1 (spike protein subunit 1, S1)和S2，S1亚基与宿主受体结合，S2亚基介导病毒膜与宿主细胞膜(或内体膜)融合，其中S2亚基的七肽重复区1 (heptad repeat1, HR1)与HR2相互作用形成六螺旋束(six-helix bundle, 6HB)，将病毒囊膜与细胞膜或内体膜拉近发生膜融合，进而将病毒基因组释放到细胞质中，完成病毒入侵过程^[15-17] (图1)。S2蛋白在不同冠状病毒中具有高度保守性，表明其结构不会因病毒的变异而发生显著改变。抑制或阻断其介导的病毒膜融合过程能够有效阻断病毒感染，是研发抗病毒药物的理想靶标^[18-19]。本文对近年来关于S2蛋白介导病毒入侵宿主细胞的作用机制以及抗病毒药物相关研究进行综述，以期为抗冠状病毒药物研发和相关疾病的预防和治疗提供参考。

1 冠状病毒入侵宿主细胞的作用机制

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1.1 冠状病毒识别的受体或黏附因子

冠状病毒入侵宿主细胞的关键在于S蛋白与宿主细胞表面受体的特异性结合。S1区域可分为N端结构域(S1 N-terminal domain, S1-NTD)和C端结构域(S1 C-terminal domain, S1-CTD)。S1-NTD和S1-CTD均可与宿主细胞受体结合，且在不同属冠状病毒中能识别特定受体，如β属鼠肝炎病毒(mouse hepatitis virus, MHV)的S1-NTD与鼠癌胚抗原相关细胞黏附分子1a (carcinoembryonic antigen-related cell adhesion molecule 1a, CEACAM1a)结合^[20]；SARS-CoV-1和SARS-CoV-2通过S1-CTD结合血管紧张素转化酶2 (angiotensin converting enzyme 2, ACE2)；MERS-CoV则通过S1-CTD结合二肽基肽酶4 (dipeptidyl peptidase 4, DPP4)。α属的HCoV-229E识别氨基肽酶N (aminopeptidase N, APN)，HCoV-NL63也以ACE2作为受体。同时，S1亚基可通过识别蛋白质或糖类等黏附因子辅助病毒在细胞表面富集，如SARS-CoV-2的S1-NTD可结合AXL受体酪氨酸激酶(AXL receptor tyrosine kinase, AXL)^[21]、低密度脂蛋白受体结构域蛋白3 (low-density lipoprotein receptor class A domain-containing protein 3, LDLRAD3)、跨膜蛋白30A (transmembrane protein 30A, TMEM30A)和C型凝集素结构域家族4成员G (C‑type lectin domain family 4 member G, CLEC4G)^[22]等蛋白质，而PEDV、BCoV等的S1-NTD则识别唾液酸及其衍生物(如Neu5Ac、Neu5,9Ac₂等)^[23-24]，这些相互作用共同增强病毒与宿主细胞的初始结合效率。此外，S蛋白经弗林蛋白酶(furin)切割后暴露C端C-end Rule (CendR)基序，该序列可以被神经纤毛蛋白1 (neuropilin 1, NRP1)结合，进一步促进SARS-CoV-2等病毒在细胞表面的附着与入侵^[25]。

1.2 冠状病毒入侵宿主细胞的方式

冠状病毒与宿主细胞受体结合后，主要通过细胞表面膜融合和内体膜融合2种途径进入宿主细胞，其S蛋白在介导病毒入侵宿主细胞过程中发挥着决定性作用。

1.2.1 细胞表面膜融合途径

与受体结合后，宿主细胞表面的蛋白酶对S蛋白的切割是触发膜融合的关键步骤，多种蛋白酶通过切割特定位点诱导S蛋白构象变化，使S蛋白从预融合(pre-fusion)构象向融合活性构象转变，随后发生结构重排。跨膜丝氨酸蛋白酶2 (transmembrane protease, serine 2, TMPRSS2)可直接切割SARS-CoV-2的S蛋白以激活其融合能力；胰蛋白酶(trypsin)和嗜热菌蛋白酶(thermolysin)能增强多种冠状病毒的膜融合效率；弗林蛋白酶则通过切割部分冠状病毒S蛋白的S1/S2位点，为后续构象重排奠定基础。对于HCoV-HKU1等β属冠状病毒，其S蛋白的S1/S2位点虽存在多碱性弗林蛋白酶切割序列，但在细胞表面途径中TMPRSS2的丝氨酸蛋白酶结构域无需催化活性即可通过与S蛋白结合辅助入侵，而其蛋白酶活性则在促进细胞-细胞融合中发挥作用^[26]。细胞表面蛋白酶切割触发S蛋白构象变化，同时激活S2区域，形成6HB结构，介导病毒包膜与细胞膜融合，完成病毒进入过程^[27]。

1.2.2 内体膜融合途径

在内体膜融合途径中，细胞网格蛋白(clathrin, CME)、小窝蛋白(caveolin, CavME)介导的内吞途径以及巨胞饮(micropinocytosis, Macropino)等方式将病毒内吞进入细胞形成内体^[28-31]。CME介导的内吞途径是冠状病毒最常见途径之一，病毒与受体结合后诱导CME在细胞膜下聚集、内陷，形成CME有被小窝，动力蛋白(dynein)通过GTP酶活性驱动有被小窝从细胞膜脱落，完成病毒内化，在此过程中，衔接蛋白2 (adaptor protein 2, AP2)与CME β亚基结合，对包被小窝的组装至关重要^[32]。研究发现，PEDV^[30]、SARS-CoV-2^[33]、HCoV-229E^[34]、BCoV^[35]、传染性支气管炎病毒(infectious bronchitis virus, IBV)^[36]等均主要通过该途径入侵宿主细胞。分选连接蛋白10 (sorting nexin 10, SNX10)促进衔接蛋白复合物2 mu 1亚基(adaptor-related protein complex 2 mu 1 subunit, AP2M1)的磷酸化，从而增强CME介导的病毒胞吞作用^[37]。此外，CME重链(clathrin heavy chain, CHC)、动力蛋白、内吞作用辅助蛋白(epsin1)等也参与该途径的调控。

与CME介导的内吞途径不同，CavME介导的内吞途径形成的囊泡进入细胞后会先形成小窝体(caveosome)，再与内体融合，其酸化过程相对缓慢，为病毒提供了独特的胞内转运微环境。HCoV-229E在成纤维细胞中可通过该途径入侵，依赖细胞膜上的CavME和脂质筏的参与，形成不依赖CME的内吞囊泡^[38]。PEDV、BCoV和PDCoV等病毒也可以通过该途径进入靶细胞^[31,35,39]。脂质筏介导的内吞途径不依赖CME和CavME，以脂质筏为平台形成内吞结构。研究表明，IBV与宿主细胞表面受体结合后会聚集于脂质筏区域，通过脂质筏的内陷形成囊泡进入细胞^[36]。SARS-CoV-2^[40]和PEDV^[31]等冠状病毒也可以通过该途径进入细胞。此外，部分冠状病毒在特定细胞中可通过Macropino途径入侵，其特征是通过细胞膜的皱褶包裹病毒颗粒，形成大型囊泡，该途径受肌动蛋白重排和信号通路调控。研究发现，PDCoV^[41]和PEDV^[42]等病毒均可通过该途径入侵靶细胞。

内吞囊泡的胞内转运与酸化是病毒释放基因组的关键环节，冠状病毒一般要经历早期内体-晚期内体-晚期内体/溶酶体阶段。病毒内化后，内吞囊泡经Rab5介导与早期内体融合，再在Rab7作用下成熟为晚期内体，V-ATP酶通过泵入H⁺使内体pH从6.0-6.5降至4.5-5.5^[43]。BCoV的转运需Rab7和Rab11参与^[35]，PEDV需要Rab7的参与^[31]，而HCoV-OC43的内体酸化依赖SNX10对V-ATP酶活性的调节^[37]。晚期内体与溶酶体融合形成晚期内体/溶酶体，内体pH值的逐步降低会触发病毒S蛋白的构象变化，同时激活组织蛋白酶切割S蛋白形成6HB，促进病毒包膜与内体膜融合，释放病毒基因组进入细胞质^[44]。这一酸化过程对感染至关重要，氯喹、氯化铵等酸化抑制剂可通过中和内体pH值，有效阻断多种冠状病毒的感染^[31,34,45]。总之，冠状病毒能够通过多种内体途径进入宿主细胞，单一靶向药物可能无法完全阻断病毒感染，这也给抗冠状病毒药物的开发带来挑战。

1.3 冠状病毒入侵途径的偏好性

不同属冠状病毒的入侵偏好性存在显著差异，这种差异主要由其S蛋白的切割活化方式和对宿主细胞蛋白酶环境的适应性决定。β属冠状病毒主要以细胞表面膜融合的途径入侵宿主细胞，同时也可以通过内体膜融合途径侵入^[46]。位于细胞质膜的丝氨酸蛋白酶(如TMPRSS2)或者外源蛋白酶(如trypsin等)会触发细胞表面膜融合途径，与内体膜融合途径相比，细胞表面膜融合途径更直接高效^[46]。例如，SARS-CoV-2在表达TMPRSS2的鼻和支气管中倾向于通过细胞表面膜融合的入胞途径^[47]。Zhu等^[47]研究发现，S蛋白S1/S2切割位点碱性氨基酸的缺失促使病毒由细胞表面膜融合向内体膜融合途径转变，并显著降低病毒的传染性。SARS-CoV-2奥密克戎株S蛋白的N679K/P681H突变增强了组织蛋白酶切割效率，显著提升内吞入侵偏好^[48]。细胞表面膜融合途径使SARS-CoV-2能够快速感染上呼吸道纤毛细胞等靶细胞，也是其实现高效空气传播和高致病性的核心分子基础。

α属冠状病毒如PEDV和PEAV等，偏好内体膜融合途径^[28,30,49]。这些病毒的S蛋白不易被细胞表面蛋白酶有效切割，因此依赖于内体膜融合途径侵入，在内体酸性环境和组织蛋白酶B/L等蛋白酶的共同作用下，S蛋白被活化并触发膜融合。然而，研究中发现在外源添加trypsin或过表达TMPRSS2和镶嵌型丝氨酸蛋白酶大片段(mosaic serine protease large-form, MSPL)时也能够促进PEDV的增殖^[50-51]，trypsin切割依赖于S2亚基^[50]，这也从另一个角度解释了PEDV为何在富含蛋白酶的肠道中易感。γ属冠状病毒(如IBV)专一通过内体膜融合途径侵入宿主细胞。IBV S蛋白缺乏TMPRSS2切割位点，需通过CME介导的内吞进入内体，经低pH环境诱导构象变化后，由组织蛋白酶切割激活膜融合^[36]。δ属冠状病毒(如PDCoV)主要通过内体膜融合途径侵入宿主细胞^[29,41,52]。Liang等^[53]研究表明，δ属冠状病毒HKU11、HKU13、PDCoV (HKU15)和HKU17的S蛋白假病毒能够通过CME介导的内吞途径进入DF-1细胞。外源性trypsin对PDCoV感染人类来源细胞无显著影响，跨膜丝氨酸蛋白酶11E (transmembrane serine protease 11E, TMPRSS11E)能够促进PDCoV通过细胞表面膜融合途径侵入细胞，同时Huh7细胞中高表达弗林蛋白酶在PDCoV复制周期的后期阶段也起着关键作用，促进病毒粒子的成熟和释放^[54]。总之，冠状病毒入侵途径的偏好性由冠状病毒S蛋白的切割方式和组织细胞的蛋白酶环境决定，这种差异也决定了其传播力、致病性以及感染的组织器官。

2 冠状病毒S蛋白的结构与功能

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2.1 冠状病毒的S1亚基结构和功能

冠状病毒S1亚基的S1-NTD和S1-CTD均以β折叠为核心。不同冠状病毒的S1-CTD呈现保守的β-折叠片层结构，SARS-CoV的S1-CTD含五链反平行β-折叠，MERS-CoV则为四链反平行β-折叠，它们通过受体结合基序(receptor binding motif, RBM)分别结合ACE2和DPP4。S1-NTD在BCoV中呈半乳糖凝集素折叠结构，形状为含糖结合口袋，可特异性识别唾液酸衍生物Neu5,9Ac₂，从而黏附于宿主细胞，而MHV的S1-NTD通过类似折叠方式识别黏附因子CEACAM1^[55]。

S蛋白未与宿主细胞受体结合时，S蛋白主要以受体结合区域(receptor binding domain, RBD) “向下”的“关闭”预融合构象存在(图2A)，由S1和S2亚基通过非共价键连接^[56-58]，这种天然构象有助于免疫逃逸。S1形成三聚体头部，S2构成茎结构，这种亚稳态结构依赖亚基间相互作用维持，可防止S蛋白过早发生构象重排^[55]。当病毒靠近宿主细胞时，1个RBD与受体结合，其余RBD会迅速转变为“向上”的开放状态构象，暴露出RBD基序，准备与受体结合^[59]。RBD与受体结合后，会触发S蛋白的构象改变。Xing等^[60]使用SARS-CoV-2的S蛋白和ACE2共培养的方法，捕捉到了一种称为早期融合中间构象(early fusion intermediate conformation, E-FIC)的激活中间构象，在这种状态下S1通过RBD-SD1与相邻的N端结构域之间的相互作用形成一个环(图2B)；与预融合S相比，S2亚基经历了显著的转变，其中HR1-中心螺旋(central helix, CH)被排出形成三股卷曲螺旋，而S2仍与S1结合，此结构的出现为后续的膜融合步骤做好准备。S蛋白三聚体中每个S1亚基的C端与S2亚基N端存在界面作用，而S2亚基的核心结构在膜融合过程中发生显著重排^[61]。

2.2 冠状病毒的S2亚基结构和功能

冠状病毒S2蛋白是其S蛋白的C端部分，负责病毒包膜与宿主细胞膜的融合过程^[42]。S2亚基包含融合肽(fusion peptide, FP)、HR1、CH、连接结构域(conserved domain, CD)、HR2、跨膜区域(transmembrane domain, TM)和胞内尾(intracellular tail, IC)等区域(图3)^[60,62]。在预融合构象中，HR1形成三聚体卷曲螺旋，HR2呈柔性状态，FP被包裹于内部。E-FIC出现以后，位于S1与S2亚基连接区域的S1/S2和S2′ 2个切割位点，经弗林蛋白酶或组织蛋白酶L等切割后触发向融合活性构象转变^[63-64]。S2亚基与S1亚基脱落形成融合后(post-fusion)构象(图2C)：S2片段中的HR1与CH形成超长中心三链卷曲螺旋(长度约218 Å)，带动原本埋藏的功能型FP (残基867-909)发生约185 Å的空间位移，插入宿主细胞膜并形成发夹状楔形三聚体贯穿脂质双层；同时HR2反向折叠回绕HR1卷曲螺旋形成稳定的6HB结构(图2D)，新增的α^{1 148-1 155}螺旋进一步加固该结构，而TM (残基1 212-1 234)倾斜包裹FP楔形三聚体，IC形成三角形结构封端膜内侧，FP邻近区域(FP proximal region, FPPR) (残基835-856)通过盐桥与FP锚定，最终S2片段形成高度刚性的三聚体终态，将病毒膜与宿主膜紧密拉近并完成融合^[62]。FP上游的857-866位片段位于脂质双层的头部基团区域，其多个疏水残基朝向膜的疏水核心，该构象可帮助相邻的二硫键锁定FPPR，使其C端半段也嵌入膜中，而FPPR的N端半段因连接无序的s^816-834片段而远离膜；此外，FPPR与FP楔形结构通过盐桥紧密结合，FPPR既在融合前协助固定RBD，也可能在融合后帮助锚定S2的膜相互作用区域^[62]。冠状病毒S2亚基的TM与IC形成的结构模块，在维持预融合构象稳定性和介导病毒颗粒组装中发挥辅助作用。TM的疏水螺旋通过与包膜脂质的相互作用锚定S蛋白，而IC的短肽序列可能参与N蛋白的相互作用，这一结构特征确保了S蛋白在病毒表面的正确定位。整体而言，S2亚基的结构既保证了预融合状态的稳定性，又能通过构象重排完成膜融合，其保守结构域为广谱抗病毒药物的设计提供了靶点^[65]。

2.2.1 6HB

6HB是S蛋白从预融合到融合后构象转变的终末构象。6HB的3个HR1螺旋通过HR重复序列的疏水相互作用形成中央卷曲螺旋，HR2随后结合到HR1链形成的侧沟中，且HR1与HR2之间存在广泛的相互作用，包括疏水相互作用和氢键相互作用^[66]。对于α属冠状病毒，CCoV-HuPn-2018的6HB整体呈左旋超螺旋，长度约为116 Å，直径30 Å，而SADS-CoV的6HB长度约107 Å，直径30 Å，两者均呈现典型的杆状结构^[66]。

HR1与HR2的结合具有高度互补性，在SARS-CoV-2中HR2的疏水残基(如V1 164、L1 166等)嵌入HR1的疏水凹槽，同时极性残基间形成特异性相互作用(如HR1的K933与HR2的N1 192、D936、R1 185分别形成2.7 Å的盐桥)，这些是维持6HB稳定性的关键^[67]。6HB结构在不同属冠状病毒中存在差异，α属的CCoV-HuPn-2018因HR1含14个氨基酸残基插入，HR2也相应延长，形成23-turn的HR1螺旋与9-turn的HR2螺旋；而SADS-CoV因缺乏氨基酸残基插入，HR1为19-turn、HR2仅5-turn，结构更接近β属；HR1三聚体中央核心由疏水氨基酸(如Leu、Ile)侧链紧密堆积形成疏水核心，HR1与HR2的对应疏水残基进一步增强6HB的结构稳定性^[66]。γ和δ冠状病毒的6HB结构核心区域高度保守，均由HR2双螺旋包裹HR1三聚体核心组成，该构象与α和β冠状病毒相同，疏水相互作用位点、氢键与盐桥等关键作用位点保守。总体而言，冠状病毒6HB的形成本质一致、核心功能相同并且基本形态相似，这也是可以通过阻断6HB的形成设计广谱抗冠状病毒药物的核心理论。

2.2.2 S2亚基的保守性

S2亚基作为冠状病毒S蛋白中高度保守的功能核心，其结构和功能区域具有高度保守性，这种保守性为广谱抗病毒策略提供了关键基础。研究表明，与变异率较高的S1亚基相比，S2的保守性在不同冠状病毒间更为显著。例如SARS-CoV与SARS-CoV-2的融合结构域具有90%的序列一致性^[68]。S2亚基的整体折叠模式在β属冠状病毒中高度一致，其核心结构域的空间排布几乎无显著差异^[69]。Wang等^[70]在S2亚基的膜近端茎螺旋(stem helix, SH)区域鉴定了一个中和抗体表位，该表位在β冠状病毒中存在功能保守性，也是自然感染(人、单峰驼感染MERS-CoV)和免疫后机体产生抗体的主要靶标之一。

S2的保守性体现在多个介导膜融合的结构域中。FP作为膜插入的关键结构，其疏水核心序列在α属和β属冠状病毒中高度保守，所有冠状病毒的S蛋白均具有高度保守的膜近端外部区域(membrane-proximal external region, MPER)和内部融合肽(internal fusion peptide, IFP)，其中IFP区域的保守性使其成为肽类模拟物药物开发的理想靶点^[71]。Ou等^[71]对β冠状病毒S2亚基的潜在FP进行研究，发现HR-N上游17-22个氨基酸的区域具备Ⅰ类病毒融合蛋白FP特征，该区域富含Ala或Gly，相对疏水且不含带电残基，对MERS-CoV (C系)、SARS-CoV (B系)、MHV (A系)的FP进行定点突变后，绝大多数疏水残基的精氨酸突变均会完全/显著抑制S蛋白介导的细胞-细胞融合和病毒入侵，GXXXG基序在所有冠状病毒的FP区域中均有分布，具有高度的保守性。S2亚基的核心结构域(HR1-CH、6HB和不变β-折叠)确保了构象重折叠的稳定性，关键功能位点(FP、TM和FPPR)确保膜融合的有效性，且保守性优先体现在结构模式和功能互作，而非全序列一致^[62]。MPER作为S2亚基靠近跨膜域的关键片段，其保守性同样显著，且该区域的胆固醇识别序列在β属冠状病毒中高度同源，参与膜融合的最终阶段^[72]。S2亚基的TM和IC虽序列较短，但空间构象在不同冠状病毒中高度一致，TM的疏水螺旋通过与包膜脂质的相互作用锚定S蛋白的机制具有保守性。Dacon等^[65]从进化角度指出，S2亚基的HR1-HR2相互作用界面在β属冠状病毒中高度保守，其氨基酸序列的一致性超过80%。

冠状病毒S2亚基的保守性也体现在其高级构象上，尤其在融合前和融合后的核心状态。冠状病毒S蛋白构象转变的基本路径为受体结合/S1脱落、S2′位点切割、FP弹出、HR1-HR2形成6HB结构，S2亚基完成预融合构象、融合中间态、稳定的融合后构象这一系列精确的构象重排过程^[16,60]。此外，S2亚基在构象转变过程中的中间态(如E-FIC)及其关键结构(如IL770环)也呈现保守特征，IL770环中S2′位点的切割机制在冠状病毒中普遍存在，是触发融合肽插入和构象重排的核心事件^[60]。6HB构象也是所有冠状病毒都具有的最终构象，这种构象的存在也使得靶向HR1或HR2区域设计广谱抗冠状病毒药物成为可能^[18-19]。

3 冠状病毒膜融合抑制剂研究

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3.1 基于HR1或HR2多肽的融合抑制剂

3.1.1 靶向HR1多肽

基于HR2序列设计的外源性多肽可竞争性结合HR1，形成稳定的6HB结构，阻断病毒自身HR1和HR2的相互作用，抑制病毒膜融合过程^[18-19]。该方案以冠状病毒HR2的氨基酸序列为模板，直接截取参与6HB形成的核心功能片段，无需引入脂化、环化、定点突变等额外结构修饰。针对SARS-CoV (如CP-1)^[73-74]、MERS-CoV (如MERS-HR2P)^[75-76]、PEDV (如PEDV HR2P)^[77]、HCoV-229E (如229E-HR2P)^[78]等多个冠状病毒的研究均取得成功。SARS-CoV-2出现以后，多个团队快速设计或筛选了HR2衍生肽，均显示出良好的抗病毒活性^[67,79-80]。然而，大多直接截取的HR2片段由于片段较短，空间构象较差，导致多肽的活性较低，一般为微摩尔级别。同时，此类多肽能够有效抑制蛋白酶介导细胞表面膜融合的病毒感染，但对内体膜融合途径的影响很小^[81]，因此需要探索新的设计策略开发抑制多种感染途径的药物，进一步提高药物的抗病毒活性。

为了增加HR2多肽与HR1区域的结合能力，提高多肽的结构稳定性和抗病毒活性，各研究团队对多肽进行了不同改造。(1) 多肽序列改变，如在HR2多肽序列中引入负电荷和正电荷的氨基酸Glu(E)和Lys(K)的多肽(EK1)^[18]、HR2多肽向N端延长(long HR2_42)以及点突变^[82-85]，均能够提高多肽的活性。(2) 环化肽，如碳氢化合物“订书钉”短α螺旋肽(P21S10、SCH2-1-20)^[86-88]。(3) 脂肽修饰，以天然HR2多肽的核心功能序列为骨架，在N端或C端引入脂溶性基团(胆固醇、棕榈酰基、硬脂酰基等)，部分通过聚乙二醇(polyethylene glycol, PEG)链或天然氨基酸连接子(如甘氨酸-丝氨酸-甘氨酸(glycine-serine-glycine, GSG))连接脂链与多肽主体，兼顾靶向结合活性与膜穿透能力。代表性的多肽如EK1C4^[67]、IPB02衍生物^[80,89-90]、HRC脂肽^[56]、EK1-C16A^[91]、P315V3 (HY3000)^[19]和[SARSHRC-PEG 4]2-chol^[92]。以上多肽改造策略均大幅提高多肽的抗病毒活性，具有良好的应用前景。

关于脂肽的研发已形成多个药物产品，正在进行临床试验。例如，EK1雾化剂于2021年9月获得国家药品监督管理局下发的临床试验批准通知书，目前处于Ⅲ期临床试验阶段^[93]。HY3000鼻喷雾剂目前在中国完成Ⅱ期临床试验(https://www.hybio.com.cn)，并于2023年1月28日获得美国食品药品监督管理局批准可进入临床研究阶段^[19]。最新研究结果显示，HY3000能够高效抑制新出现的SARS-CoV-2 EG.5、EG.5.1和BA.2.86变异株^[94]，目前也处于Ⅲ期临床试验阶段。源于IPB02衍生物肽的脂肽药物YKYY017雾化吸入剂，Ⅱ期随机对照试验结果显示，YKYY017干预轻度至中度新冠患者，吸入20 mg YKYY017的患者在第4天的病毒载量减少量高于安慰剂，显示出潜在的抗病毒活性，吸入10 mg和20 mg YKYY017耐受性良好，安全性可接受^[95]。雾化吸入IPB29也已经进入Ⅲ期临床试验阶段^[96]。以上进入临床试验阶段的冠状病毒HR2多肽类药物，为后续开发靶向HR2的药物提供了强大的理论和研发路径支撑。

3.1.2 其他类型的HR1多肽衍生物

为了进一步突破天然多肽在稳定性、半衰期、靶向性及生物利用度等方面的固有限制，不同团队进行了多肽衍生物的制备，包括引入非天然结构单元、功能结构域融合、纳米材料修饰或多聚化组装等方案。(1) 非天然骨架修饰：如以磺酰-γ-AA等非天然氨基酸替代天然L-氨基酸，改变肽链空间构象，降低蛋白酶识别位点暴露，同时维持与HR1的疏水结合活性，提升口服生物利用度^[97]。(2) 功能结构域融合多为双靶点融合设计，如将红藻凝集素G (griffithsin, GRFT)^[98]、纤维连接蛋白Ⅲ型结构域(fibronectin type III domain (FN3)，可与白蛋白结合)^[99]、ACE2或ACE2衍生的A1肽段^[60,100-101]以及RBD区域^[102]等与HR2衍生肽连接，形成新的蛋白分子，与单独使用各组分相比，均能大大提升其抗病毒活性，其中RBD-IPB01蛋白展现出广谱性、低细胞毒性、高胰蛋白酶抵抗性和良好代谢稳定性^[102]。(3) 纳米化修饰，如将金纳米棒等载体通过疏水作用与多肽结合，保护肽链不被降解，同时提升细胞穿透效率，改善生物相容性^[103]。总之，上述HR1或HR2多肽衍生物从多个层面对药物进行改造或修饰，以期提高其功能活性或成药性，为抗冠状病毒多肽类药物开发提供参考，同时探寻提高多肽药物的吸收和代谢的新方法也是未来研究的重点。

3.1.3 靶向HR2的多肽

在冠状病毒S蛋白三聚体融合前结构中HR1区域被包裹埋藏，因此理论上基于HR2设计的HR1靶向肽，仅在S蛋白融合中间体形成时发挥功能，而HR2位于外围，也是设计阻断病毒膜融合的靶标。Sun等^[104]靶向MERS-CoV HR2区域设计了一个包含3个HR1和2个HR2的蛋白MERS-5HB，该蛋白与HR2区域稳定结合，有效抑制MERS-CoV假病毒进入细胞，以及显著抑制S蛋白介导的膜融合。Xing等^[105]靶向SARS-CoV-2 HR2区域设计了5HB蛋白，该蛋白能与HR2区域形成稳定的6HB结构，有效抑制SARS-CoV-2野生型毒株(nCoV-SH01)及其Delta变异株的感染。此外，Lin等^[106]也设计了5HB，结果显示该蛋白能够在血清和低pH环境中与SARS-CoV-2 S蛋白融合前结构结合，暗示其在SARS-CoV-2通过细胞膜表面或内体膜融合途径进入时的抑制能力，同时该蛋白也能够中和S介导的主要SARS-CoV-2变异株及多种沙粒病毒科(Arenaviridae)的感染。总之，5HB设计方案也可以作为一种制备冠状病毒新型抑制剂的策略。

3.2 靶向FP区域的多肽

除了靶向HR1和HR2区域以外，还有团队靶向FP区域设计了多肽分子。Safiriyu等^[107]基于冠状病毒的FP区域设计了多肽MFP633，该多肽含有中央脯氨酸-脯氨酸二联体，能够抑制MHV感染和复制，利用脂质体介导递送系统将多肽给予小鼠，其表现出良好的抗病毒活性。此类研究报道相对较少，还处于前期研究阶段。

3.3 抗体类抑制剂

S蛋白是冠状病毒的主要免疫原性蛋白，在诱导机体产生免疫保护中发挥关键作用。针对S2区域筛选抗冠状病毒的抗体也是开发抗病毒药物的热点。本课题组对PEDV的S1-COE (CO-26K equivalent)区域进行了表达和纯化，该区域是PEDV的一个重要中和表位区域，并被用作设计PEDV亚单位疫苗^[108]。本课题组通过免疫小鼠制备单克隆抗体，并对其抗原表位进行测定，最终筛选得到一个新的线性B细胞表位-⁵⁹²TSLLASACTIDLFGYP⁶⁰⁷，该表位的最短识别序列为⁶⁰⁴FGYP⁶⁰⁷，其中604F、605G和606Y为其关键识别位点；保守性分析结果显示，该表位在PEDV各毒株中高度保守^[109]。该表位的鉴定进一步丰富了PEDV的抗原表位谱，为PEDV亚单位疫苗的设计提供参考。此外，靶向SARS-CoV-2 S蛋白RBD的全人源IgG1单克隆抗体贝特洛韦(bebtelovimab)，能够有效中和Omicron多种变异株，其作用机制为直接干扰病毒进入宿主细胞，降低病毒载量^[110]。安巴韦单抗/罗米司韦单抗(Amubarvimab/Romlusevimab)是我国首次获批的具有自主知识产权的中和抗体联合治疗SARS-CoV-2药物，在我国COVID-19患者的Ⅱ期临床研究中表现出良好的安全性、耐受性及临床获益趋势^[111]。

目前报道的抗体类药物主要靶向S蛋白的RBD，此外针对S2亚基的抗体也逐渐受到关注^[112]。Pinto等^[113]研究发现，S2亚基茎螺旋区域的单克隆抗体S2P6与多种β冠状病毒S蛋白的SH交叉反应，通过与该区域的疏水侧结合阻碍6HB形成，抑制病毒膜融合；S2P6通过病毒中和及Fc介导效应功能，减轻感染SARS-CoV-2的仓鼠的病毒负荷。Sun等^[114]从一名SARS-CoV-2康复患者中分离得到76E1抗体，该抗体靶向S2′位点和FP，阻断病毒膜融合的发生。Dacon等^[65]也筛选到靶向S2′位点和融合肽的广谱抗冠状病毒的中和抗体。Ko等^[115]通过mRNA-LNP免疫策略筛选获得b-S2-Mab-2抗体，其可以靶向S2亚基HR2的D1 146-F1 152保守序列，对多个SARS-CoV-2变异株具有良好的中和活性。靶向S2亚基的纳米抗体S102^[116]和抗体54043-5^[117]等也显示出良好的抗病毒活性。此外，Pang等^[118]靶向S2亚基HR1结构域设计了HR121蛋白疫苗，该蛋白能够诱导兔子和恒河猴产生SARS-CoV-2及其变异株，尤其是Omicron亚谱系的高效交叉中和抗体，同时能够保护hACE2转基因小鼠、叙利亚金仓鼠和恒河猴抵抗SARS-CoV-2原始毒株的感染，可以作为抗SARS-CoV-2变异毒株的疫苗候选。可见，靶向S2亚基区域的抗体也显示出良好的抗病毒活性，也是抗体筛选和疫苗制备的优秀靶标。

3.4 其他类型的抑制剂

其他类型的抑制剂中，小分子化合物、微小RNA(microRNA, miRNA)分子及老药新用策略均展现出显著的冠状病毒膜融合抑制活性。Yang等^[119]基于SARS-CoV-2 S2亚基筛选小分子膜融合抑制剂，发现丹酚酸C特异性结合HR1区域，有效抑制S蛋白介导的膜融合、假病毒感染，同时能够抑制SARS-CoV-2活病毒感染。万欣等^[120]研究发现，马铃薯三糖甘草次酸衍生物与S1与S2亚基交界处的空腔结合，对SARS-CoV-2奥密克戎假病毒有显著抑制作用，同时对其他SARS-CoV-2变异株假病毒有广谱抗病毒活性。miRNA分子方面，Vaddadi等^[121]发现多种靶向SARS-CoV-2 S蛋白的miRNA，能够将S蛋白水平降低超过50%，显著降低病毒复制。老药新用策略中，阿比多尔(Arbidol)是一种俄罗斯制造的抗病毒药物，用于治疗某些包膜病毒和非包膜病毒。一项临床随机对照试验显示，阿比多尔显著促进SARS-CoV-2患者的临床和实验室检测指标的改善^[122]。研究表明，阿比多尔可以通过阻碍病毒的附着和从细胞内囊泡释放发挥抗SARS-CoV-2的作用^[123]。Shuster等^[124]研究表明，阿比多尔与SARS-CoV-2 S2亚基膜融合结构域(1 046-1 066位氨基酸残基)结合，抑制病毒入侵细胞并诱导S蛋白的溶酶体降解。以上抑制剂可以为抗病毒治疗提供潜在替代方案，以及与多肽或抗体类药物联用，提高抗病毒效果。

4 总结与展望

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冠状病毒S2蛋白介导的膜融合是病毒入侵的关键环节，也是抗病毒药物研发的重要靶点。近年来，随着对冠状病毒S2蛋白介导膜融合机制的深入研究，越来越多的关键位点和结构域被揭示，尤其是多款靶向SARS-CoV-2 S2亚基的喷鼻或吸入式药物进入临床研究阶段，这些研究成果为抗冠状病毒药物研发提供了重要依据和思路。冠状病毒具有高度变异性和抗药性，这使得针对其S2蛋白的药物研发面临巨大挑战：S2蛋白构象变化的动态性和复杂性给结构解析和机制研究带来困难；药物靶点的筛选与验证需要建立更高效的评估体系；病毒变异可能产生的耐药性需要前瞻性考虑；抑制剂的体内递送和稳定性问题亟待解决。

靶向冠状病毒S2亚基的抗病毒药物的未来研究方向可能包括：开发靶向S2蛋白不同功能域(FP、HR1、HR2、TM等)的多靶点抑制剂，寻找高结合能力、高广谱性的多肽、抗体药物或化合物等，以“鸡尾酒”的形式制备抗病毒药物，增强其抗病毒活性，延长其耐药性；利用人工智能辅助药物设计和优化，能够高效地设计抗病毒药物^[125]，借助机器学习算法高效解析冠状病毒S2蛋白动态构象变化、精准预测药物与FP、HR1、HR2等功能域的结合位点，同时可前瞻性模拟病毒变异对靶点结构的影响，辅助设计出更易适配变异株的药物分子，大幅提升药物研发效率与针对性；探索新型递送系统(如纳米颗粒、外泌体等)，尤其适合肠道冠状病毒的给药方式，有效保护抑制剂免受体内酶解，增强靶向性以精准抵达感染部位，同时延长药物循环时间，显著改善抑制剂的体内稳定性与生物利用度，破解当前抑制剂递送效率低、易被清除的难题；研究S2蛋白与宿主因子的相互作用，发现新的宿主靶点分子，明确其与S2蛋白的作用特征，阐明其分子作用机制，为开发靶向宿主分子抗病毒药物提供参考。

总之，设计出能够应对病毒变异和抗药性的抗冠状病毒药物是保障公共卫生安全的重要课题。随着多学科交叉融合和技术创新，针对冠状病毒膜融合过程的干预策略将不断丰富和完善，为应对当前和未来冠状病毒威胁提供有力武器。特别是基于S2蛋白高度保守性开发的广谱抑制剂和疫苗，有望成为预防和控制冠状病毒大流行的关键手段。

基金

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中国博士后科学基金(2023M741084)
国家自然科学基金(32102687)

参考文献

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2026年第66卷第7期

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doi: 10.13343/j.cnki.wsxb.20250828

接收时间：2025-11-03
首发时间：2026-07-06
出版时间：2026-07-04

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收稿日期：2025-11-03
录用日期：2026-03-01

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The China Postdoctoral Science Foundation(2023M741084)

中国博士后科学基金(2023M741084)

The National Natural Science Foundation of China(32102687)

国家自然科学基金(32102687)

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河南中医药大学医学院，河南郑州

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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