AVN-944

中国病毒学英文版 IF:4.7 公众号ID：virologica 关注 阅读原文 https://doi.org/10.1016/j.virs.2026.07.009 作者  孙倩，刘克森，曹万迪，吴承月，张汉华，王兴娅，彭辰，孙洁，张安兵，周卓，刘星 单位 a深圳大学医学部药学院 b深圳大学医学部，基础医学院，广东省感染免疫与炎症重点实验室 c中国农业大学兽医学院兽医公共卫生国家重点实验室 d深圳海关动植物检验检疫技术中心 e中山人民医院呼吸与危重症医学科 f中国医学科学院&北京协和医学院，苏州系统医学研究所，重大疾病共同机制研究国家重点实验室 摘要      猴痘病毒(MPXV)作为一种高致病性正痘病毒，已引发全球性疫情，构成严重的公共卫生威胁。目前，用于治疗天花的抗病毒药物虽已被应用于猴痘感染的临床干预，但其应用受到药物储备有限及耐药株出现等因素的制约。病毒通过重塑宿主核苷酸代谢网络，获取复制与传播所必需的生物合成前体，该机制已成为开发广谱抗病毒药物的重要靶向策略。本研究利用痘苗病毒(VACV)与猴痘病毒在遗传和生物学特性上的高度相似性，以VACV为替代模型，系统筛选了10种美国FDA批准的靶向核苷酸代谢酶的抑制剂，以期发现新型猴痘病毒抑制候选药物。结果显示，吗替麦考酚酯(MMF)-一种肌苷酸5'-脱氢酶Ⅱ型(IMPDH2)抑制剂，对VACV和MPXV均表现出显著的抗病毒活性。进一步的药物作用时相分析表明，MMF主要干预病毒的复制阶段。机制研究揭示，MMF可阻断泛素特异性蛋白酶5(USP5)介导的IMPDH2去泛素化过程，并且能诱导细胞中出现“rod-and-ring”(R&R)结构，进而抑制IMPDH2功能，降低细胞内dNTP池水平，最终有效抑制猴痘病毒的复制。综上所述，本研究证实MMF是一种高效的抗痘苗病毒及猴痘病毒药物，同时建立了一种以宿主因子为靶点的抗病毒治疗策略，为应对未来猴痘疫情暴发提供了新的干预思路。 Fig. 1. Screening of nucleotide metabolism inhibitors and evaluation of antiviral efficacy. A Schematic diagram of the nucleotide biosynthesis pathways and key enzymes targeted by inhibitors. B Overview of the compound screening workflow. HeLa cells were cultured overnight in 96-well plates, then treated with 10 µM candidate compounds, and incubated for 24 h under CO₂ conditions. After incubation, fluorescence intensity was recorded using an automated scanning system to assess antiviral activity. C HeLa cells were treated with 10 µM drugs from panel A and infected with A5-GFP at an MOI of 0.1 for 24 hours. Cytarabine (Ara- C) was used as the positive control compound. GFP fluorescence was visualized using a fluorescence microscope. Scale bar: 100 µm. D Quantification of GFP fluorescence intensity from panel C. Relative fluorescence values were normalized to cell number. E–G HeLa cells were treated with 10 µM drugs and infected with A5-GFP at an MOI of 0.1 for 24 hours. Dose–response curves showing antiviral efficacy (EC₅₀) and cytotoxicity (CC₅₀) of (E) Mycophenolic acid (MPA), (F) Mycophenolate mofetil (MMF), and (G) AVN-944. The upper green curves represent antiviral activity, while the lower red curves indicate cytotoxicity. (mean ± SD of n = 3 biological replicates in panels C–G). ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Fig. 2. MMF inhibits poxvirus replication. A-B Cells were infected with VACV and MPXV at an MOI of 0.1 for 24 hours, and simultaneously treated with 10 µM MMF or DMSO. Viral titers in the HeLa cells (A) or A549 cells (B) were measured by plaque assay. C Schematic representation of the experimental design used to evaluate the effects of MMF on different stages of the viral life cycle. D-E HeLa cells were treated with 10 μM MMF or DMSO and subsequently infected with VACV (D) or MPXV (E). Viral binding, entry, and replication were quantified by qPCR, whereas viral release was assessed by plaque assay. (mean ± SD of n = 3 biological replicates in panels A, B, D and E). ns, not significant, ***P < 0.001. Fig. 3. MMF targets IMPDH2 to inhibit viral replication. A Relative IMPDH2 mRNA levels quantified by qRT-PCR in control and IMPDH2-knockdown HeLa cells. B–C Wild-type and IMPDH2-knockdown HeLa cells (B) and control and IMPDH2-overexpressing HeLa cells (C), were infected with VACV at an MOI of 0.1 for 24 h and treated with 10 μM MMF or DMSO. Viral titers were measured by plaque assay. D HeLa cells were mock-infected or infected with VACV at an MOI of 0.1 and treated with 10 µM MMF or DMSO for 24 h, after which cell lysates were harvested to quantify IMPDH2 levels using an ELISA kit. E HeLa cells were treated with 10 µM MMF or DMSO, heated at indicated temperatures, and analyzed by Western blot using an antibody against IMPDH2, with GAPDH serving as a control. The thermal shift curve shows normalized IMPDH2 protein levels. Quantification of IMPDH2 band intensity at 50°C from three independent CETSA experiments. F HeLa cells were treated with increasing concentrations of guanosine (0–1000 µM) for 24 h. Cell viability was measured by CCK-8 assay. G HeLa cells were infected with VACV at an MOI of 0.1 and treated with 10 µM MMF, and then treated increasing concentrations of guanosine for 24 h. Viral titers were measured by plaque assay. H Wild-type and IMPDH2 knockdown of HeLa cells were infected with VACV at an MOI of 0.1 and treated 1000 µM guanosine or DMSO for 24 h. Viral titers were measured by plaque assay. I MMF is converted to its active form, mycophenolic acid (MPA), via esterase-mediated hydrolysis. J IMPDH2 (pink) structure in complex with MPA (orange). Magnified views show hydrogen bonds (yellow dashes, distances in Å) between MPA and residues Ser276, Gly326, Thr333, and Gln441. Right panel shows 90° rotated view of the binding site. (mean ± SD of n = 3 biological replicates in panels A–F). ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Fig. 4. USP5-mediated deubiquitination of IMPDH2 facilitates viral replication. A Volcano plots showing differentially expressed genes comparing mock-infected or infected. B HeLa cells were infected with VACV at an MOI of 0.1, and cell samples were collected at 12 and 24 hours, respectively. Total RNA was extracted and subjected to RT-qPCR analysis for IMPDH2 mRNA expression. C Volcano plot of quantitative proteomic analysis depicting differentially expressed proteins upon VACV mock-infection or infection. D HeLa cells seeded in 12-well plates were infected at an MOI of 0.1. At 24 hours post-transfection (hpt), cells were lysed for immunoblotting with antibodies against IMPDH2, B5 and GAPDH. E Relative USP5 mRNA levels in control or USP5 knockdown HeLa cells. F Wild-type and USP5 knockdown of HeLa cells were either mock-infected or infected with VACV at an MOI of 0.1. At 24hpt, cells were lysed for immunoblotting with antibodies against IMPDH2,USP5,B5 and GAPDH. G HeLa cells were either mock-infected or infected with VACV at an MOI of 0.1 for 24h, and proximity ligation assay (PLA) was performed with anti‑USP5 and anti‑IMPDH2 antibodies to detect USP5-IMPDH2 interaction., Scale bar: 10 µm. H HeLa cells seeded in 6-cm dishes were either mock-infected or infected with VACV. At 24 hpi, cell lysates were immunoprecipitated with a rabbit anti-IMPDH2 antibody or control IgG, followed by western blot analysis with against USP5, IMPDH2, B5 and GAPDH. I Wild-type and USP5 knockdown of HeLa cells seeded in 6-cm dishes were either mock-infected or infected with VACV and treated with the proteasome inhibitor MG132 (10 µM). At 24 hpi, cell lysates were immunoprecipitated with a rabbit anti-IMPDH2 antibody, followed by western blot analysis with against UB, USP5, IMPDH2, B5 and GAPDH. J Wild-type and USP5 knockdown of HeLa cells were either mock-infected or infected with VACV at an MOI of 0.1 for 24 h, after which cell lysates were harvested to quantify IMPDH2 levels using an ELISA kit. K Wild-type or USP5-knockdown of HeLa cells were infected with VACV at an MOI of 0.1 for 24 h. Viral titers were measured by plaque assay. L-O HeLa cells were mock-infected or infected with VACV at an MOI of 0.1 and treated with 10 µM MMF or DMSO for 24 h. L Proximity ligation assay (PLA) was performed with anti‑USP5 and anti‑IMPDH2 antibodies to detect USP5-IMPDH2 interaction., Scale bar: 10 µm. M Cell lysates were subjected to western blot analysis using antibodies against USP5, IMPDH2, B5, GAPDH. N Cells were subjected to immunoprecipitation with a rabbit anti-IMPDH2 antibody or control IgG. Whole-cell lysates (WCLs) and immunoprecipitated proteins were analyzed by immunoblotting with antibodies against IMPDH2, USP5, B5 and GAPDH. O HeLa cells were treated with the proteasome inhibitor MG132 (10 µM) and then cells were subjected to immunoprecipitation with a rabbit anti-IMPDH2 antibody. WCLs and immunoprecipitated proteins were analyzed by immunoblotting with antibodies against UB, IMPDH2, USP5, B5 and GAPDH. (Data are representative of at least three independent experiments. mean ± SD of n = 3 biological replicates in panels A, C, F and I-K). ns, not significant, ***P < 0. 001. Fig.5. MMF inhibits viral replication by inducing IMPDH2 Rod and Ring assembly, thereby inhibiting its enzymatic activity. A Schematic model illustrating stepwise assembly of IMPDH2 from monomers into higher-order oligomers and Rod and Ring (R&R) filaments. B-D HeLa cells were either mock-infected or infected with VACV at an MOI of 0.1 and treated with 10 µM MMF or DMSO. B At 24 hpt, cell lysates were resolved by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) or standard SDS-PAGE, followed by immunoblotting using antibodies against IMPDH2, B5 and GAPDH. C Cells were fixed and stained for IMPDH2 (red), VACV (green) and nuclei (DAPI, blue), and imaged using confocal microscopy. Scale bars: 10 µm. D The quantity of IMPDH2 filaments in each cell was measured as in panel C, with at least 100 cells analyzed per group. E IMPDH2 knockdown cells overexpress the drug-binding site mutant and wild-type of IMPDH2 were treated 10 µM MMF, and imaged using confocal microscopy. Scale bars: 10 µm. F The quantity of IMPDH2 filaments in each cell was measured (panel D and panel F), with at least 100 cells analyzed per group. G IMPDH2 knockdown cells overexpress the drug-binding site mutant and wild-type of IMPDH2 for 24 hours, after which cell lysates were harvested to quantify IMPDH2 levels using an ELISA kit. H IMPDH2 knockdown cells overexpress the drug-binding site mutant and wild-type of IMPDH2. At 12hpt, cells were treated with 10 µM MMF or DMSO, after which cell lysates were harvested to quantify IMPDH2 levels using an ELISA kit. I Cells used the same treatment conditions as in panel H. Viral titers were measured by plaque assay. (mean ± SD of n = 3 biological replicates in panels C-I). ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Fig. 6. MMF treatment prolonged the survival of VACV-infected mice and reduced viral loads in multiple organs. A Schematic of the experimental design. Six-week-old female BALB/c mice (n=12 per group) were divided into four groups. Mice were either mock-infected or infected with 1×10⁴ PFU of VACV. B Survival analysis. Mouse survival was monitored for 14 days according to the design in panel A. C Body weight changes. The body weight of mice was monitored daily for 14 days and is presented as the mean percentage of initial weight. D-E Viral load quantification. Viral loads in the lungs (D) and brains (E) were measured by qPCR at 6 days post-infection (dpi). F-G Histopathological analysis. Tissues for analysis were collected from mice that were euthanized at 6 days post-infection (dpi). Representative hematoxylin and eosin (H&E)-stained sections of lung (F) and brain (G) tissues were collected at the experimental endpoint. Scale bars: 100 µm. Data are representative of at least three independent experiments with similar results (mean ± SD, n = 8 biological replicates in panels B and C, n = 4 biological replicates in panels D and E). *** P < 0.001. 本文亮点 吗替麦考酚酯（MMF）通过靶向IMPDH2抑制痘病毒感染 MMF可抑制USP5介导的IMPDH2去泛素化修饰，进而下调IMPDH2酶活性 MMF 能够诱导 IMPDH2 形成 “rod-and-ring”(R&R)” 结构，从而抑制其酶活性 相关阅读： 清华大学丁强团队：布鲁顿酪氨酸激酶抑制剂BTKi-2抑制猴痘病毒和痘苗病毒感染 | VS推荐 Bruton's tyrosine kinase inhibitor BTKi-2 inhibits mpox virus and vaccinia virus infection 痘病毒的跨物种传播与宿主范围基因 | VS推荐 Cross-species transmission and host range genes in poxviruses 一株新型鼠痘病毒的分离鉴定推动疫苗和抗病毒药物研究 | VS推荐 Identification of a novel ectromelia virus from rodent: Implications for use as an in vivo infection model for vaccine and antiviral research 猴痘病毒竟有"偏好通道"！科学家解密免疫缺陷小鼠易感性差异| VS推荐 Differential susceptibility of immunodeficient mice to MPXV infection and the impact of various inoculation routes The unique immune evasion mechanisms of the mpox virus and their implication for developing new vaccines and immunotherapies Isolation and characterization of mpox virus from the first mpox case in Shenzhen, China VS 往期直达 2026-2 2026-1 2025-6 2025-5 2025-4 2025-3 2025-2 2025-1 2024-6 2024-5 2024-4 2024-3 期刊简介 《中国病毒学（英文）》Virologica Sinica, 是中国科学院武汉病毒研究所和中国微生物学会共同主办的病毒学领域的专业学术期刊。聚焦病毒发现及流行规律、病毒致病机理、病毒-宿主互作、疫苗及抗病毒药物研发、病毒相关生物技术等领域，覆盖病毒学全链条研究。本刊最新影响因子4.7，5年影响因子4.6, 均位于病毒学领域前15%(JCR2025), CiteScore 2025 7.3, 连续十二年入选“中国最具国际影响力学术期刊” (TOP 5%)。期刊于2022年变更为以金色开放获取模式出版的开源期刊（Open Access Journal），与科爱出版社合作全球出版传播。本刊为中国科技核心期刊，且被SCI、PubMed/Medline、PubMed Central、Scopus等数据库收录。