人腺病毒B种7型（Human adenovirus Species B Type 7，HAdV-B7）是儿童急性下呼吸道感染（acute lower respiratory tract infections，ALRTIs）的主要病原体之一，严重威胁儿童健康。2009~2020年我国急性下呼吸道感染暴发疫情中有48.71%是由HAdV-B7引起的。国内外研究表明，HAdV-B7 感染与重症肺炎、中毒性脑病、弥散性血管内凝血（disseminated intravascular coagulation，DIC）、急性呼吸窘迫综合征（acute respiratory distress syndrome，ARDS）等严重的临床综合征密切相关，与HAdV-B其他型别相比更易引起重症病例，甚至死亡。但是，HAdV 感染致病机制不明，且国内尚无疫苗或特效药。因此，病毒复制过程中宿主因子与病毒复杂的相互作用成为研究抗病毒药物靶点的重要方向，亟需深入探究HAdV-B7感染的病毒-宿主相互作用机制，寻找潜在药物靶点，为研发特效抗 HAdV-B7 药物提供新策略。

本研究旨在探究调控 HAdV-B7 复制的关键宿主蛋白，寻找潜在的抗病毒药物靶点，并阐明该宿主因子调控 HAdV-B7 复制的分子机制。基于高通量筛选策略，本研究在HeLa细胞上过表达人类 15000 个 cDNA 文库质粒，然后感染 HAdV-B7 临床分离株，并借助免疫荧光的方式在高内涵显微成像系统，获得调控HAdV-B7 复制的候选宿主蛋白。然后通过 siRNA 敲降、CRISPR/Cas9 敲除、回补实验以及小分子抑制剂等处理，结合免疫印迹法、实时荧光定量 PCR 分析（quantitative real-time PCR，qRT-PCR）、免疫荧光、TCID₅₀ 等方法检测病毒蛋白、转录本以及滴度变化，验证候选宿主因子调控病毒复制的作用表型。再进一步通过内化结合实验、突变体实验、高效液相串联质谱分析（liquid chromatography-tandem mass spectrometry，LC-MS/MS）、AlphaFold2 蛋白结构预测、蛋白-蛋白分子对接、免疫共沉淀（co-immunoprecipitation，co-IP）、截断体实验等，明确 HAdV-B7 感染过程中的病毒-宿主相互作用机制。此外，本研究还通过多重序列对比和最大似然法进化树分析等探究了候选宿主因子对于不同型别 HAdV 作用差异的理论依据。

通过两轮高通量筛选和验证实验，本研究发现糖原合成激酶 3 α（glycogen synthase 3 α, GSK3A）是促进 HAdV-B7 感染复制的关键宿主因子。细胞实验表明，HAdV-B7 感染后 32 和 48 h 可观察到 GSK3A 蛋白及转录本表达上调。过表达外源性 GSK3A 促进 HAdV-B7 复制。GSK3A 敲降或敲除后 HAdV-B7 复制水平显著下降，回补 GSK3A 后病毒复制恢复。GSK3 小分子抑制剂 CHIR99021 显著抑制 HAdV-B7 病毒复制。广谱性研究表明，GSK3A 敲除后 HAdV-B~E均受到不同程度地抑制，其中HAdV-B作用最为显著。机制上，内化结合实验表明 GSK3A 敲除不影响 HAdV-B7 进入过程。qRT-PCR 分析表明在 GSK3A 敲除细胞中，病毒基因 E1A、Hexon、DBP 转录本水平在 HAdV-B7 感染后 8 h 出现显著性下调，提示 GSK3A 缺失抑制病毒转录。HAdV-B7 感染后蛋白激酶 B （protein kinase B，AKT）丝氨酸 473 位点（Serine 473，S473）磷酸化和 GSK3A S21 磷酸化水平下调，提示 GSK3A 激酶活性持续激活。突变体实验表明，GSK3A 的促病毒作用依赖其自身激酶活性。磷酸化失活位点 GSK3A-S21A 突变后 HAdV-B7 复制增强；磷酸化激活位点 GSK3A-K148A 和 Y279A 突变则抑制 HAdV-B7 复制。磷酸化蛋白质组学和免疫印迹分析发现 GSK3A 的促病毒作用不依赖自噬、干扰素应答、WNT/β-catenin 通路等，而是通过调控病毒蛋白磷酸化谱实现，包括 8 个蛋白的 13 个磷酸化修饰肽段在 GSK3A 缺失后显著下调。免疫共沉淀实验表明，GSK3A 能够与多种病毒蛋白如 L4-22K、E1A-I、DBP 等互作，并促进 L4-22K 磷酸化水平。随后的截断体实验表明 GSK3A 利用其激酶结构域结合病毒 L4-22K 蛋白 274~504 aa 区域，调控 L4-22K S78 和 S81 位点磷酸化修饰，促进 HAdV-B7 病毒复制。序列对比和进化树分析表明，L4-22K 蛋白 S78 位点在 HAdV-B 中高度保守，S81 位点在 HAdV-B3 中保守，提示L4-22K 的 SXXS 基序在不同 HAdV 中的保守性与病毒在 GSK3A 缺失后受到抑制的敏感性有关。

本研究通过高通量功能获得性筛选和功能验证实验，揭示了丝氨酸/苏氨酸激酶 GSK3A，而非 GSK3B，在 HAdV-B7 和 HAdV-B3 复制中具有关键调控作用。机制研究表明，GSK3A 依赖其激酶活性促进病毒复制和转录；GSK3A 缺失使 8 个病毒蛋白的 13 个磷酸化修饰肽段丰度下调，从全局角度改变了病毒蛋白的磷酸化修饰谱。截断体实验表明，GSK3A 利用激酶结构域结合 L4-22K 蛋白，调控其 S78 和 S81 磷酸化修饰，促进病毒复制。在抗病毒治疗方面，本研究验证了 GSK3 小分子抑制剂 CHIR99021 的抗病毒作用，为临床进一步开发或推动特异性 HAdV-B7 抗病毒药物提供了靶点和小分子化合物的线索。综上，本研究阐明了丝氨酸/苏氨酸激酶 GSK3A 在 HAdV-B7 感染中的宿主-病毒相互作用机制以及其型别特异性，揭示了病毒挟持宿主细胞内激酶，激活病毒蛋白自身磷酸化修饰协助病毒完成复制的分子机制，深化了对 HAdV-B7 感染的表观遗传调控机制的理解。

Human adenovirus species B type 7 (HAdV-B7) is one of the major pathogens associated with acute lower respiratory tract infections (ALRTIs) in children, posing a serious threat to child health. Between 2009 and 2020, 48.71% of the acute lower respiratory infection outbreaks in China were attributed to HAdV-B7. Studies at home and abroad have shown that HAdV-B7 infection was closely associated with severe clinical syndromes, including severe pneumonia, toxic encephalopathy, disseminated intravascular coagulation (DIC), and acute respiratory distress syndrome (ARDS). Compared to other types of HAdV-B, it is more likely to result in severe cases, including fatalities. However, the pathogenic mechanisms of HAdV infection remain unclear, and no vaccines or effective drugs are currently available. Therefore, the intricate interactions between host factors and the virus during viral replication have become a significant focus for developing antiviral drug targets. There is an urgent requirement for further investigates on the virus-host interaction mechanisms in HAdV-B7 infection to identify potential therapeutic targets, providing new strategies for developing effective treatments against HAdV-B7.

This study aims to identify key host proteins that regulate HAdV-B7 replication, seek potential antiviral drug targets, and elucidate the molecular mechanisms through which these host factors modulate HAdV-B7 replication. Utilizing a high-throughput screening strategy, we overexpressed a human cDNA library of 15,000 plasmids in HeLa cells, followed by infection with clinical isolate HAdV-B7 strains. The candidate host proteins involved in regulating HAdV-B7 replication were identified through immunofluorescence using a high-content imaging system. Subsequent experiments involved siRNA knockdown, CRISPR/Cas9 knockout, complementation assays, and small molecule inhibitors to assess changes in viral proteins, viral gene transcripts, and viral titers using methods such as immunoblotting, quantitative real-time PCR (qRT-PCR), immunofluorescence, and TCID₅₀ assays to verify the phenotypic effects of candidate host factors on viral replication levels. Further investigation into the virus-host interaction mechanisms during HAdV-B7 infection involved internalization assays, mutant experiments, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, AlphaFold2 protein structure predictions, protein-protein molecular docking, co-immunoprecipitation (co-IP), and truncation studies.

Through two rounds of high-throughput screening and subsequent validation experiments, our research identified glycogen synthase 3 alpha (GSK3A) as a key host factor that enhances HAdV-B7 infection and replication. Cell experiments demonstrated that GSK3A protein and transcript levels were upregulated after HAdV-B7 infection at 32 and 48 h respectively. Overexpression of exogenous GSK3A promoted HAdV-B7 replication, while GSK3A knockdown or knockout resulted in a significant decrease in HAdV-B7 replication levels. Complementation with GSK3A restored viral replication, and the small molecule inhibitor CHIR99021 significantly inhibited HAdV-B7 replication. Broad-spectrum studies revealed that knocking out GSK3A hindered members of HAdV-B, HAdV-C, and HAdV-E to varying degrees, with the most significant effect observed in HAdV-B.

Mechanistically, internalization assays indicated that GSK3A knockout did not affect HAdV-B7 entry. qRT-PCR analysis showed significant downregulation of viral genes E1A, Hexon, and DBP transcripts in GSK3A knockout cells at 8 hours post-infection, suggesting that GSK3A is crucial for viral transcription. Following HAdV-B7 infection, we observed downregulation in phosphorylation levels of protein kinase B (AKT) at serine 473 (S473) and GSK3A at S21, indicating sustained activation of GSK3A kinase activity. Mutant experiments revealed that the pro-viral activity of GSK3A is dependent on its kinase activity. The phosphorylation-inactive mutant GSK3A-S21A enhanced HAdV-B7 replication, while phosphorylation-activated mutants GSK3A-K148A and Y279A inhibited viral replication. Phosphoproteomic and Western blot analyses discovered that the pro-viral activity of GSK3A does not rely on autophagy, interferon response, or WNT/β-catenin pathway, but rather regulates the phosphorylation profile of viral proteins, with 13 phosphorylated peptides from 8 proteins significantly downregulated upon GSK3A deletion. Co-immunoprecipitation experiments indicated that GSK3A interacts with multiple viral proteins, such as L4-22K, E1A-I, and DBP, and promotes the phosphorylation level of L4-22K. Subsequent truncation experiments demonstrated that GSK3A utilizes its kinase domain to bind to the 274-504 aa region of the viral L4-22K protein, regulating the phosphorylation modifications at S78 and S81, thus promoting HAdV-B7 replication. Sequence comparisons and phylogenetic tree analyses suggested that the S78 site of L4-22K protein is highly conserved within HAdV-B, while the S81 site is conserved in HAdV-B3, indicating the conservation of the SXXS motif of L4-22K across different HAdV types may relate to the sensitivity of viral replication to GSK3A depletion.

This study reveals that the serine/threonine kinase GSK3A, rather than GSK3B, plays a crucial regulatory role in the replication of HAdV-B7 and HAdV-B3, based on high-throughput functional screening and validation experiments. Mechanistic studies indicate that GSK3A promotes viral replication and transcription in a kinase activity-dependent manner. The absence of GSK3A downregulates the abundance of 13 phosphorylated peptide modifications in 8 viral proteins, thereby altering the phosphorylation profile of viral proteins on a global scale. Truncation experiments confirmed that GSK3A binds to L4-22K protein via its kinase domain, regulating its phosphorylation modifications at S78 and S81 to promote viral replication. In terms of antiviral therapy, our research validates the antiviral effects of the GSK3 small molecule inhibitor CHIR99021, providing insights into potential drug targets and small molecule compounds for the further clinical development of specific antiviral therapies targeting HAdV-B7. In summary, this research elucidates the virus-host interaction mechanisms of the serine/threonine kinase GSK3A in HAdV-B7 infection and its type-specific characteristics, revealing the molecular mechanism by which the virus hijacks host cell kinases to activate its own phosphorylation modifications, facilitating viral replication and enhancing our understanding of the epigenetic regulatory mechanisms in HAdV-B7 infection.