在宿主抵御病原体入侵的初始应答阶段，先天免疫系统通过模式识别受体（pattern recognition receptors，PRRs）精确识别病原体保守分子特征，迅速激活胞内信号级联反应。这一过程最终诱导 I 型干扰素及其下游效应分子的表达。其中，鸟苷酸结合蛋白（guanylate-binding protein， GBP）作为干扰素应答的核心效应分子，其抗感染功能依赖于其 N 端 GTP 酶结构域的 GTP 水解能力和 C 端效应结构域的膜锚定能力，二者协同介导其抗病原体效应的实现。然而，该家族诸多成员在天然免疫中的具体功能及其调控机制尚不清晰。已有研究表明，维甲酸诱导基因-I-线粒体抗病毒信号蛋白（retinoic acid-inducible gene I- mitochondrial antiviral signaling protein，RIG-I-MAVS）信号传导抑制 H5N1 病毒的复制过程依赖于GBP1，但 GBP 家族是否参与调控环鸟苷酸-腺苷酸合成酶-干扰素基因刺激蛋白（cyclic GMP-AMP synthase-stimulator of interferon genes，cGAS-STING）通路仍缺乏相关研究。本研究基于初期筛选结果，聚焦于 GBP 家族成员 GBP4，深入探究其在 STING 介导的 I 型干扰素（IFN-I）通路中的调控作用及其对病毒复制的影响，旨在揭示 GBP4 在天然免疫与病毒感染中的潜在功能与机制。

本研究首先证实了 IFNα、IFNβ、IFNγ均可诱导 GBP 家族成员的转录表达。随后，构建了 GBP 家族各成员的表达质粒，并与 STING 共同转染细胞，检测IFNB的转录水平。结果显示在所有 GBP 成员中，GBP4 在 STING 介导的 IFN-I应答中表现为最为显著的促进作用。为了进一步探究 GBP4 的作用， 本研究采用剂量梯度方式过表达 GBP4，同时转染 STING，实验结果表明，GBP4 呈剂量依赖性增强 STING 信号通路的活化，具体表现为 TANK 结合激酶 1 （TANK-binding kinase 1，TBK1）、干扰素调节因子 3（interferon regulatory factor 3，IRF3）及STING 的磷酸化水平上调，同时下游干扰素相关基因的表达显著增加。 鉴于GBP家族蛋白的多种功能依赖其 GTP 酶活性，本研究推测 GBP4 促进 STING 通路的活化的作用亦可能依赖其酶活性结构域。为验证该假设， 本研究构建了缺失 GTP酶功能关键区域的 GBP4 突变体：GBP4-M1 （Δ60-67）、 GBP4-M2 （Δ82-84）和GBP4-M3（Δ112-116），将其分别与 STING 共转染。结果显示，GBP4-M2 显著削弱甚至抑制了 STING 通路的活化， 提示 GBP4 增强 STING 信号通路的活化依赖其 GTP 酶活性结构域（82-84 位氨基酸）。

为进一步阐明 GBP4 调控 cGAS-STING 信号通路的机制，本研究通过免疫荧光实验观察到 GBP4 定位于细胞胞浆，且与 STING 呈显著共定位。免疫共沉淀实验进一步表明，GBP4 通过其 N 端和 C 端结构域与 STING 的环二核苷酸结合结构域（CDN-binding domain，CBD）相互作用，且在 STING 激动剂 diABZI刺激下，STING 与 GBP4 的互作显著增强，这一结果提示 GBP4 可能通过促进STING 寡聚化或调节其亚细胞定位以增强其信号传导。与此同时，GBP4 缺失GTP 酶功能关键区域的三个突变体均与 STING 存在相互作用， 说明 GBP4 的 82–84 区域对 STING 通路活性的调控可能依赖于其 GTP 酶功能，而非其与 STING的结合能力，两者功能相互独立。

基于 GBP4 对 STING 介导的 I 型干扰素信号通路的调控作用，本研究进一步分析了其在宿主抗病毒过程中的功能。 首先 GBP4 可以在多种病毒感染下被诱导表达。此外，本研究进一步评估了 GBP4 在 DNA 病毒感染及复制中的功能。结果显示， GBP4 的缺失显著促进了痘苗病毒（Vaccinia Virus， VACV） 的复制，提示 GBP4 可能作为 VACV 的负向调控因子。而在 I 型单纯疱疹病毒（herpes simplex virus 1，HSV-1）感染中，GBP4 的过表达促进其复制，GBP4 的敲除则抑制了 HSV-1 蛋白的表达。值得注意的是，即使在使用 JAK 抑制剂 Ruxolitinib阻断 Janus 激酶-信号转导和转录激活因子（Janus kinase-signal transducer and activator of transcription，JAK-STAT）通路后，GBP4 敲除细胞中 HSV-1 复制仍被抑制，提示 GBP4 促进 HSV-1 复制的作用独立于经典的干扰素信号通路。

综上所述，本研究系统揭示了 GBP4 在天然免疫应答中的关键作用， 首次明确其可通过增强 STING 信号通路活化，促进 I 型干扰素及下游抗病毒因子的表达，且该功能依赖其 GTP 酶活性结构域。同时，GBP4 与 STING 在细胞质内共定位并直接相互作用，提示其可能通过调控 STING 的构象状态或亚细胞分布实现信号增强。进一步研究发现，GBP4 在不同 DNA 病毒感染中的功能存在显著差异：GBP4 抑制 VACV 的复制，却促进 HSV-1 的复制，且后者作用不依赖于经典 JAK-STAT 通路。这些结果不仅揭示了 GBP4 在病毒感染中所发挥的多样调控作用，也为深入理解 GBP 蛋白家族在天然免疫调控中的功能及其作为干预靶点的可行性提供了新思路。

During the early stages of host defense against pathogen invasion, the innate immune system rapidly recognizes conserved microbial signatures through pattern recognition receptors (PRRs), initiating intracellular signaling cascades that ultimately induce type I interferons (IFN-I) and downstream effector molecules. Guanylate-binding proteins (GBPs), key effectors of interferon responses, exert their antimicrobial functions through the coordinated action of their N-terminal GTPase domain—which mediates GTP hydrolysis-and the C-terminal effector domain responsible for membrane anchoring. However, the precise functions and regulatory mechanisms of many GBP family members in innate immunity remain poorly understood. Previous studies have shown that inhibition of H5N1 replication via the RIG-I-MAVS pathway depends on GBP1. Whether GBPs also participate in the regulation of the cGAS-STING signaling axis remains unclear.

Based on initial screening results, this study focuses on GBP4, investigating its regulatory role in the STING-mediated IFN-I signaling pathway and its impact on viral replication, with the aim of elucidating its function and mechanism in innate immunity and viral infection. We first confirmed that IFN-α, IFN-β, and IFN-γ induce transcription of various GBP family members. Expression plasmids for individual GBPs were constructed and co-transfected with STING to assess IFNB transcription levels. Among all GBPs tested, GBP4 exhibited the most pronounced enhancement of STING-mediated IFN-I signaling. Further analysis showed that overexpression of GBP4 enhanced STING signaling in a dose-dependent manner, as indicated by increased phosphorylation of TBK1, IRF3, and STING, along with elevated expression of downstream interferon-stimulated genes. Given the known importance of GTPase activity for GBP function, we hypothesized that GBP4’s effect on STING activation might depend on its enzymatic domain. To test this, we generated three GBP4 mutants lacking key GTPase residues—GBP4-M1 (Δ60–67), GBP4-M2 (Δ82–84), and GBP4-M3 (Δ112–116)—and co-transfected them with STING. Notably, GBP4-M2 significantly impaired STING activation, suggesting that residues 82–84 in the GTPase domain are critical for GBP4’s regulatory function.

Immunofluorescence revealed that GBP4 is localized in the cytoplasm and colocalizes with STING. Co-immunoprecipitation experiments further demonstrated that GBP4 interacts with the cyclic dinucleotide-binding domain (CBD) of STING via both its N- and C-terminal regions. This interaction was markedly enhanced by the STING agonist diABZI, implying that GBP4 may promote STING oligomerization or modulate its subcellular localization to enhance signal transduction. Importantly, all three GTPase-deficient mutants retained the ability to bind STING, indicating that the regulatory effect of the 82–84 region on STING activation is mediated by GTPase activity rather than binding affinity, suggesting distinct and independent functional domains.

To assess the antiviral role of GBP4, we first confirmed that it is inducible upon infection by various viruses. Functional studies revealed that GBP4 deficiency significantly enhanced replication of Vaccinia virus (VACV), suggesting it acts as a negative regulator of VACV. In contrast, GBP4 overexpression promoted replication of herpes simplex virus 1 (HSV-1), while GBP4 knockout suppressed HSV-1 protein expression. Notably, the pro-viral effect of GBP4 on HSV-1 persisted even in the presence of the JAK inhibitor Ruxolitinib, indicating that this function is independent of the classical JAK-STAT signaling pathway.

In conclusion, this study reveals a critical role for GBP4 in innate immunity, demonstrating for the first time that it enhances IFN-I responses by activating the STING pathway in a GTPase-dependent manner. GBP4 colocalizes and directly interacts with STING, potentially modulating its conformation or localization to facilitate signaling. Furthermore, GBP4 exerts divergent effects on DNA viruses, inhibiting VACV replication while promoting HSV-1 replication via a JAK-STAT–independent mechanism. These findings not only highlight the versatile regulatory roles of GBP4 in antiviral responses but also offer new insights into the functional diversity of GBPs and their therapeutic targeting in innate immunity.