综述：病毒复制与磷酸肌醇动态的相互调控
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时间：2025年10月15日
来源：FEBS Letters 3
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本综述系统阐述了磷酸肌醇（PIPs）在病毒复制与宿主免疫中的双重作用机制，揭示了其作为（PI4P）、（PI(4,5)P2）等关键脂质分子如何被病毒劫持以构建复制细胞器（ROs），同时调控（TLRs）、（RIG-I）、（cGAS-STING）等天然免疫通路。文章深入探讨了靶向（PI4Ks）、（OSBP）等宿主因子的广谱抗病毒策略，为开发新型宿主导向疗法提供了重要理论依据。
磷酸肌醇（PIPs）作为细胞中稀少但高度动态的脂质分子，在膜动力学、细胞内运输和信号转导中起核心调控作用。病毒作为专性细胞内病原体，已进化出劫持宿主细胞机制的策略，包括脂质代谢通路，以支持其复制。在病毒感染过程中，病毒利用PIPs完成其周期的多个步骤，包括进入、复制细胞器形成、组装和释放。
PIPs是膜运输、细胞骨架动力学和内吞作用的关键调节因子，这些过程均被病毒利用以进入宿主细胞。值得注意的是，磷脂酰肌醇-4,5-二磷酸（PI(4,5)P2）是一种关键的脂质支架，通过招募AP-2复合物、网格蛋白和切割因子来成核网格蛋白介导的内吞作用，这是许多病毒如甲型流感病毒（IAV）和登革病毒（DENV）进入的常见途径。在人类免疫缺陷病毒-1（HIV-1）的情况下，病毒进入涉及融合孔的形成，由T细胞表面糖蛋白CD4受体和趋化因子共受体[C-X-C趋化因子受体4型（CXCR4）和C-C趋化因子受体5型（CCR5）]的聚集启动。这种聚集事件诱导激酶磷脂酰肌醇4-磷酸5-激酶I型α（PIP5K-Iα）的激活，从而产生PI(4,5)P2。同时，HIV-1糖蛋白gp120在病毒与宿主细胞接触期间也触发PIP5K-Iα活性，从而增加PI(4,5)P2水平。
PIPs也是埃博拉病毒（EBOV）进入宿主细胞的关键调节因子。脂质磷脂酰肌醇3,5-二磷酸（PI(3,5)P2）由激酶1-磷脂酰肌醇3-磷酸5-激酶（PIKfyve）产生，对于内体成熟和EBOV递送至含有尼曼匹克C1（NPC1；也称为NPC细胞内胆固醇转运蛋白1）受体的晚期内体至关重要，这是病毒融合的关键步骤。
PIPs，尤其是磷脂酰肌醇4-磷酸（PI4P），在病毒复制区室的形成和维持中起核心作用。这些区室，也称为复制细胞器（ROs），是许多正链RNA（+RNA）病毒依赖的特殊膜结构，用于高效基因组复制。ROs的形态和复杂性因其病毒来源而异；例如，丙型肝炎病毒（HCV）感染期间产生双膜囊泡（DMVs），而登革病毒（DENV）或寨卡病毒（ZIKV）等黄病毒则诱导源于内质网（ER）内陷的囊泡包（VPs）。
病毒诱导的宿主细胞膜重塑以创建这些复制平台涉及结构重排和特定宿主脂质信号通路的招募， notably those involving PI4P. For example, DMVs formed during HCV infection contain significantly elevated levels of PI4P. This lipid enrichment is achieved through the hijacking of host phosphatidylinositol 4-kinase-IIIα (PI4KIIIα/PI4KA), which is recruited and activated by HCV nonstructural protein 5A (NS5A), and PI4KIIIβ/PI4KB.
PI4P在此背景下的主要作用之一是通过招募脂质转移蛋白（如氧化固醇结合蛋白（OSBP））促进关键脂质交换过程。OSBP介导胆固醇从内质网到高尔基体或病毒复制细胞器的转移，以交换PI4P。通过在其复制位点产生PI4P，病毒增强OSBP招募，从而促进胆固醇流入，这是膜刚性、曲率和病毒复制的关键脂质成分。抑制PI4KB或OSBP会破坏这种脂质运输通路，导致病毒RNA合成减少和复制细胞器结构 disorganization。
除了OSBP，其他PI4P结合蛋白在复制细胞器的功能中也起重要作用。其中一个蛋白是四磷酸衔接蛋白2（FAPP2；也称为pleckstrin homology domain-containing family A member 8），它定位于高尔基体，参与糖鞘脂运输。FAPP2与HCV复制有关，其耗竭显著破坏病毒RNA复制。另一个关键参与者是神经酰胺转移蛋白（CERT），它对于神经酰胺（鞘磷脂（SM）的前体）的运输至关重要，这是一种在HCV感染期间对DMVs的结构和功能至关重要的脂质。
值得注意的是，一些病毒也利用内吞系统获取复制所需的胆固醇。例如，PI4K不仅产生PI4P，还通过与回收内体相关的小GTP酶Ras相关蛋白Rab11相互作用参与运输功能。Rab11阳性内体可以被招募到复制细胞器以促进脂质递送。此外，几个晚期内体固醇结合蛋白，包括StAR相关脂质转移蛋白3（STARD3）和NPC1，将低密度脂蛋白（LDL）衍生的固醇转移到病毒复制细胞器中，并已被确定为高效HCV复制所必需。
总之，PI4P在病毒重编程宿主脂质代谢以形成和维持复制区室的策略中起重要作用。病毒刺激PI4K活性以在其复制位点产生PI4P，进而驱动脂质转移蛋白（如OSBP和FAPP2）的招募，并与其他区室建立接触位点。这种相互连接的网络提供脂质，如胆固醇，用于复制细胞器的构建和功能。通过协同利用这种机制，病毒创建一个受保护的环境，支持高水平基因组复制，同时逃避宿主免疫检测。
令人惊讶的是，尽管黄病毒（如DENV或ZIKV）的复制严重依赖内质网脂质重塑并利用宿主脂质通路，但OSBP循环的蛋白质，包括VAP、Sac1和PITPs，以及PI4Ks，迄今为止并未被显著涉及。然而，药理学研究表明，OSBP抑制剂阻断DENV和ZIKV复制，表明OSBP可能发挥某些作用，但缺乏进一步的机制证据。此外，一项无偏蛋白质组学筛选将OSBP相关蛋白ORP9和ORP11置于日本脑炎病毒（JEV）和ZIKV-NS5相互作用组中，表明它们在正黄病毒感染期间被招募。最近的一份报告表明，ORP9和ORP11在内质网-反式高尔基网络（TGN）接触位点形成异源二聚体，反向交换磷脂酰丝氨酸（PS）和PI4P。有趣的是， resulting PS buildup in the Golgi apparatus promotes the synthesis of SM, an essential lipid for the flavivirus lifecycle. Finally, PI4P itself could play a role in ZIKV infection by facilitating RO formation: a study shows that nonstructural protein 1 (NS1) has a high affinity for PI4P and that this interaction promotes NS1-induced ER remodeling. Downregulating PI4P levels impairs NS1-induced ER remodeling and attenuates ZIKV replication.
PIPs在病毒颗粒组装和释放过程中占据中心 stage across very different viruses, including HIV-1 and EBOV. In the case of HIV-1, which assembles and buds at the plasma membrane (PM) of infected cells, PI(4,5)P2 plays a dual role, both as a PM-targeting signal and as a functional component of the viral assembly platform. First, PI(4,5)P2 directs the Gag polyprotein to the PM via specific binding to its matrix (MA) domain. Then, Gag multimerization reorganizes the lipid environment, trapping PI(4,5)P2 and cholesterol into rigid membrane nanodomains, which facilitate virus budding. Finally, Gag-driven local lipid remodeling selectively enriches PI(4,5)P2 in the viral envelope during assembly.
在EBOV的情况下，基质蛋白VP40直接结合PI(4,5)P2以及PS via two basic, lysine-rich regions located on its C-terminal domain (CTD). Specifically, PS primarily interacts with CTD region 1, facilitating the initial association of VP40 with the PM and inducing conformational changes necessary for oligomerization. However, the stability of these oligomers is limited when only PS is present. PI(4,5)P2 interacts more strongly with CTD region 2, leading to much greater stabilization of VP40 oligomers and dimers. PI(4,5)P2 binding results in highly stable oligomeric structures, which are critical for efficient virus-like particle formation and budding. Notably, decreasing PI(4,5)P2 levels or preventing VP40–PI(4,5)P2 binding impairs filamentous virion formation.
PIPs also play a key role in coordinating the last stages of the viral cycle of IAV, a segmented negative-stranded RNA virus. Upon nucleus exit, IAV viral ribonucleoproteins (vRNPs) must travel to the PM. Recent studies indicate that vRNPs are transported through the cytoplasm by first associating with a remodeled ER, then being loaded onto irregularly coated vesicles that bud from the ER. These vesicles, which rely on the host factor RAB11A, ferry the vRNPs to the PM. Interestingly, IAV co-opts the host protein autophagy-related protein 16-1 (ATG16L1) at ER subdomains, where it likely acts as a scaffold or regulator, facilitating the local synthesis of PI4P by interacting with Hyccin (FAM126A; a subunit of the PI4KA complex) and inositol polyphosphate 5-phosphatase K (INPP5K; a 5-P phosphatase that generates PI4P). This PI4P enrichment creates specialized membrane platforms that recruit RAB11A and its effectors, driving membrane remodeling and the formation of vRNP-coated transport vesicles. Of note, RAB11A also regulates PI4P turnover and distribution, suggesting that this coordinated action ensures efficient trafficking of viral components to the PM. At the PM, PI(4,5)P2-rich nanodomains facilitate the recruitment and clustering of IAV matrix (M1) and hemagglutinin (HA) proteins, promoting coordinated membrane curvature and budding. Super-resolution studies showed that PI(4,5)P2 not only marks assembly sites but also contributes to membrane reorganization during virion scission.
Similarly, nonsegmented negative-strand RNA viruses from the Paramyxoviridae family, which includes measles and Nipah virus, use PI(4,5)P2 to coordinate their budding from the PM of infected cells. In this case, PI(4,5)P2 acts as a crucial membrane anchor and regulator of the viral matrix (M) proteins. At assembly sites beneath the PM, M proteins form a paracrystalline lattice that bridges viral glycoproteins with the ribonucleocapsid complex, driving virion formation. It was shown that M binds specifically to PI(4,5)P2, and depletion or inhibition of this lipid reduces M PM localization, virus-like particle production, and virus replication. Moreover, PI(4,5)P2 binding induces conformational rearrangements in M dimers that expose a basic patch, enhancing contacts with PM lipids, such as PS. Functionally, PI(4,5)P2 is indispensable for M-driven membrane deformation and for M oligomerization into lattices on the membrane.
PIPs such as PI4P and PI3P are also exploited during the complex egress process of the Poxiviridae vaccinia virus (VACV). Following initial assembly in cytoplasmic factories, VACV virions are wrapped in crescent-shaped membrane bilayers derived from the ER, forming spherical immature virus (IV) upon closure. These IVs are subsequently enveloped with membrane cisternae derived from the Golgi or endosomal compartments, giving rise to mature virus (MV). A set of conserved viral proteins called virus membrane-associated proteins (VMAPs) is actively involved in this process and is essential for viral membrane biogenesis and the generation of infectious progeny. Studies have shown that MVs are enriched in PIPs and that several PI3Ks are involved in VACV morphogenesis, including the IV-to-MV transition, likely by enriching these membranes with PI3P. Interestingly, the VMAP H7 possesses a putative PX domain involved in binding PI3P and PI4P. This interaction is essential for the formation of IV and the infectious brick-shaped MV, as H7 drives proper formation of hexamers of the scaffold protein D13, which shapes the newly assembled viral membrane into a sphere by organizing a honeycomb-like lattice.
During HCV infection, rewired PI4P also plays an essential role in Golgi phosphoprotein 3 (GOLPH3)-mediated viral secretion: PI4P generated by PI4KB at the TGN recruits GOLPH3, which in turn interacts with unconventional myosin-XVIIIa (MYO18A) to exert a tensile force on budding vesicles. Disrupting PI4KB, GOLPH3, or MYO18A impaired the trafficking of HCV particles through the Golgi without affecting viral RNA replication, indicating that this PI4P–GOLPH3–MYO18A axis specifically supports HCV assembly and egress.
细胞天然免疫过程负责检测病毒并参与适应性免疫反应的激活。允许这些过程中涉及的通路启动的一个关键步骤是宿主受体对病毒成分的识别和信号级联的诱导。这导致I型干扰素（IFN）和具有广谱抗病毒活性的细胞因子的产生和分泌。与其他信号通路一样，PIPs通常在感知和调节控制细胞反应的过程中起关键作用。
抗病毒反应始于模式识别受体（PRRs）超家族的天然免疫受体，包括Toll样受体（TLRs）、RIG-I样受体（RLRs）和cGAS，检测保守的病毒分子 motif，称为病原体相关分子模式（PAMPs）。PAMPs consist of virus-derived structures, such as glycoproteins, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), and unmethylated CpG-containing DNA. Upon activation, PRRs initiate signaling cascades that lead to the activation of downstream effectors, such as TANK-binding kinase 1 (TBK1; also known as serine/threonine-protein kinase TBK1), through post-translational modifications, ultimately resulting in the nuclear translocation of interferon regulatory factor 3 (IRF-3) or nuclear factor kappa B (NF-κB) to drive the expression of antiviral genes.
TLRs是I型跨膜蛋白，包含N末端细胞外/腔内富亮氨酸重复序列（LRRs）和C末端胞浆Toll/白细胞介素-1受体（TIR）结构域。PAMPs such as viral glycoproteins can be recognized by LRRs of TLR2 and TLR4 at the PM, whereas within endolysosomal compartments, TLR3 detects dsRNA, TLR7 and TLR8 sense ssRNA, and TLR9 responds to unmethylated CpG DNA. For example, TLR4 is activated by glycoproteins from several RNA viruses, such as DENV, SARS-CoV-2, EBOV, respiratory syncytial virus (RSV), and vesicular stomatitis virus (VSV).
一些PIPs帮助调节TLRs的空间定位，从而控制下游信号级联的命运。Upon sensing, TLR dimerization activates signaling pathways that originate from the interaction between TIR domains and adaptor proteins, such as TIR domain-containing adaptor protein (TIRAP) at the PM or TRIF-related adapter molecule (TRAM) at endosomes: TIRAP recruits myeloid differentiation primary response 88 (MyD88), triggering NF-κB and mitogen-activated protein kinase (MAPK) pathways, whereas TRAM is required for the engagement of TIR domain-containing adapter-inducing IFN-β [TRIF; also known as TIR domain-containing adapter molecule 1 (TICAM1)], promoting type-I IFN signaling. Of note, TIRAP encompasses an N-terminal PI(4,5)P2-binding site, which enables its recruitment to the PM and is necessary to induce proper cytokine production following TLR4 activation. PIP5Kα appears to provide the PI(4,5)P2 needed for this process, as its knockdown or inactivation in microglial cells blocked TLR4 signaling. Furthermore, degradation of PI(4,5)P2 by 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2) has been proposed to act as a molecular switch in the transition from the TLR4–TIRAP–MyD88 complex at the PM to the MyD88-independent pathway involving the TLR4–TRAM–TRIF complex at the endosomes, after internalization of TLR4 through clathrin- and dynamin-mediated endocytosis.
除了其在PM中的作用，TIRAP也被提议介导TLR7和TLR9信号传导：在缺乏TIRAP的细胞中，对TLR7和TLR9配体的反应受损，除非配体以非常高的浓度存在。为了促进MyD88与内体TLR9的 proper bridging，TIRAP被显示优先结合脂质，如PI3P、PS和可能的PI(3,5)P2，而不是PI(4,5)P2，突出了PIPs在塑造 distinct TLR信号通路中的多样化作用。
宿主细胞感知dsRNA的另一种方式是通过RLR。最近的研究揭示了PIPs在调节内体上的RIG-I信号传导中的重要作用。Upon RNA recognition, RIG-I translocates to endosomes/lysosomes, where it undergoes lysine 63 (K63)-linked ubiquitination by the E3-ligase tripartite motif-containing protein 25 (TRIM25; also known as E3 ubiquitin/ISG15 ligase TRIM25), a process that depends on the adaptor protein TBK1-associated protein in endolysosomes (TAPE; also known as coiled-coil and C2 domain-containing protein 1A). TAPE binds strongly to PI4P- or PI3P-rich membranes, enabling its endosomal attachment, and links RIG-I to TRIM25. TAPE also associates with mitochondrial antiviral-signaling protein [MAVS; also known as interferon beta promoter stimulator protein 1 (IPS-1)], a mitochondria transmembrane protein. Upon ubiquitination, RIG-I oligomerizes and binds to MAVS, triggering MAVS oligomerization. These MAVS filaments on the mitochondrial surface serve as scaffolds for recruiting downstream signaling proteins, including TBK1. Disrupting endosomal function or TAPE expression impairs ubiquitination of RIG-I and dampens antiviral responses, highlighting the importance of PIP-rich endosomal domains in supporting RIG-I signaling.
有趣的是，IAV诱导RIG-I依赖的PI3K信号传导，确保下游通路的适当激活和I型IFN的表达。PI3K activation is reported to be necessary for complete IRF-3 phosphorylation, and inhibiting either PI3K or PIP3 signaling in IAV-infected cells resulted in reduced TBK1 and IRF3 activities. Notably, IAV viral RNA (vRNA)-induced PI3K activation is abolished when RIG-I, TRIM25, and MAVS are silenced, supporting the idea that IAV vRNA serves as a stimulus for the RIG-I–MAVS axis, mediating the activation of PI3K. Inhibition of PI3K with LY29400 also decreases downstream IRF3 activation and IFNB mRNA expression following vRNA stimulation, suggesting the importance of RIG-I-dependent activation of PI3K signaling in immune response. Another study further supported a role for PI3K, along with Akt, in the regulation of IRF3 activation and IFNB expression in RIG-I–MAVS signaling upon polyinosinic-polycytidylic acid [poly(I:C)] stimulation or Sendai virus infection. Moreover, co-immunoprecipitation experiments showed that both the Akt pleckstrin homology (PH) domain and PI3K regulatory subunit p85β bind to the MAVS caspase activation and recruitment (CARD) domain. PI3K and Akt were hence suggested to associate with MAVS upon viral infection, providing a signaling platform for activation of downstream immune events. In a parallel pathway, the IAV NS1 protein directly interacts with the p85β subunit of PI3K, leading to PI3K activation and contributing to a delay in virus-induced apoptosis.
总体而言，尽管PIPs的 involvement 由其下游增强IRF3激活和I型IFN产生所暗示，但它们在调节RIG-I–MAVS复合物中的确切作用仍不清楚。类似地，PI3K信号网络对抗病毒天然免疫的贡献需要进一步阐明。鉴于其广泛参与 diverse cellular processes，PI3K激活的下游通路和生物学效应可能因时空和 context-dependent manner 而异。额外的限制包括这些研究中使用的PI3K抑制剂的潜在脱靶效应，以及PI3K在RIG-I信号传导过程中如何被接合的可能的病毒和细胞类型特异性差异。
当cGAS检测到胞浆中的病毒或自身来源的dsDNA时，cGAS-STING通路被激活，导致第二信使2′,3′-环GMP-AMP（cGAMP）的产生。然后，cGAMP与STING（一种ER驻留跨膜蛋白）的结合触发STING激活并诱导其运输到高尔基体，在那里它通过静电相互作用与管腔硫酸化糖胺聚糖（sGAGs）结合，促进其聚合。此后，聚合的STING招募并激活激酶TBK1，从而导致IRF3或NF-κB的核转位，以驱动抗病毒基因的表达。值得注意的是，cGAS通过与PI(4,5)P2的相互作用在其 inactive state 下被隔离在PM上，这种定位被证明对于防止自身DNA的不适当激活同时仍然允许有效检测病毒DNA至关重要。证据表明，STING在几种RNA病毒（如DENV、ZIKV、HCV、SARS-CoV-2和IAV）感染期间的免疫反应中也起作用。
最近的工作阐明了脂质稳态，尤其是PI4P和胆固醇，在调节STING定位和激活中的关键作用。Fang et al. showed that PI4KB and PI4P are essential for STING downstream signaling, with armadillo-like helical domain-containing protein 3 (ARMH3; C10orf76/DGARM) acting as an important mediator. ARMH3, an ADP-ribosylation factor-like protein 5 (ARL5) effector localized to the distal Golgi/TGN, recruits PI4KB, enhances PI4KB-dependent PI4P synthesis, and was found to interact with STING. The interaction between ARMH3 and PI4KB was found to be crucial for promoting STING polymerization, and, notably, only the catalytically active form of PI4KB supported STING puncta formation and downstream signaling. Moreover, the depletion of PI4P by directing Sac1 phosphatase to the TGN suppressed STING puncta formation and type-I IFN induction, emphasizing the essential role of PI4P in this pathway. Mechanistically, PI4P is thought to: (a) facilitate anterograde transport of STING from the TGN to endosomes by recruiting PI4P-binding clathrin adaptors, such as the AP-1 complex or Golgi-localized γ ear-containing ARF-binding protein 2 (GGA2), and (b) promote cholesterol and SM enrichment at the TGN by recruiting lipid-transfer proteins, thereby creating a lipid environment favorable for STING activation. The post-Golgi trafficking of STING is important for sustaining and amplifying STING activation, likely because its interaction with sGAGs is enhanced in the acidic environment of endosomes. Moreover, lipid-transfer proteins that bind PI4P, including CERT, OSBP and various OSBP-related proteins (ORP1, ORP6, ORP9, and ORP11), were found to be critical for STING activation. Indeed, the inhibition of CERT by HPA-12 or by deleting SM synthetase 1 (which converts ceramide to SM) significantly impaired STING signaling, highlighting that SM, rather than ceramide, is important for STING activation. Similarly, blocking OSBP using itraconazole (ITZ), or interfering with cholesterol trafficking using methyl-β-cyclodextrin, disrupted STING activation, indicating that cholesterol homeostasis also accounts for STING function. Furthermore, evidence suggests that STING clustering, which strengthens its association with TBK1, depends on both its palmitoylation and the ordered lipid environment of the Golgi membrane, defined by cholesterol accumulation. Notably, blocking STING palmitoylation significantly impairs TBK1 phosphorylation. Interestingly, STING activation also induces the expression of cholesterol 25-hydroxylase (CH25H), which produces 25-hydroxycholesterol (25-HC), a natural inhibitor of OSBP. By blocking OSBP, 25-HC acts as a negative regulator of STING, revealing a cholesterol-dependent feedback loop. Finally, the Golgi adaptor protein acyl-coenzyme A binding domain-containing protein 3 (ACBD3; also known as Golgi resident protein GCP60), known to recruit PI4KB to the TGN and increase its activity, was found to be another key player in STING activation during innate immune responses. ACBD3 and STING were found to colocalize in Golgi clusters and, upon ACBD3 depletion or knockout, STING remained at the ER. However, in this study, inhibition of PI4KB and OSBP had opposite effects on STING signaling: ITZ, which inhibits OSBP and thereby increases PI4P levels at the TGN, was shown to enhance STING activation. OSBP inhibition via ITZ also induced a significant decrease in STING degradation, up to 20 h after its activation.
因此，STING作为抗病毒免疫的关键参与者出现，其活性和空间调节受到脂质环境的严格控制，最 notably through PI4P accumulation at the distal Golgi, which appears to be a dominant factor in its activation. However, these observations were established essentially upon DNA stimulation and not upon RNA or poly(I:C) stimulation, which mimic RNA virus infection. Still, STING's noncanonical involvement in viral replication was recently described for some RNA viruses, either through enhancement of their replication, such as during human rhinovirus, enterovirus D68, and SARS-CoV-2 infections, in which STING localizes to PI4P-rich replication organelles, or its impairment, in the case of human coronavirus OC43 (HCoV-OC43), in which STING seems to disrupt the interaction between nonstructural proteins Nsp4 and Nsp6. In addition, the involvement of STING in the life cycle of RNA viruses of concern, notably through immune activation or evasion strategies employed by these viruses to thrive, has been discussed previously.
总体而言，PIPs不仅仅是膜组织脂质；它们可以作为区室化免疫信号传导的看门人，在定义空间定位和关键抗病毒效应物（如TLRs、RIG-I、cGAS或STING）
