{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:54:31Z","timestamp":1785430471831,"version":"3.56.0"},"reference-count":26,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T00:00:00Z","timestamp":1777334400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Guangdong Science and Technology Program key projects","award":["2021B1212030014"],"award-info":[{"award-number":["2021B1212030014"]}]},{"name":"Guangdong Special Support Plan \u201cProvincial Health Commission (Health Talents) Project\u201d","award":["0720240122"],"award-info":[{"award-number":["0720240122"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82371846"],"award-info":[{"award-number":["82371846"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["82371846"],"award-info":[{"award-number":["82371846"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Guangzhou Basic Research Program","award":["2023A03J0810"],"award-info":[{"award-number":["2023A03J0810"]}]},{"name":"Guangdong Province Basic and Applied Basic Research Fund Project","award":["2024A1515010995"],"award-info":[{"award-number":["2024A1515010995"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>The membrane fusion process, mediated by the entry fusion complex (EFC) of the monkeypox virus (MPXV), is crucial for host cell invasion. Apolipoprotein B mRNA Editing Catalytic Polypeptide-like 3 (APOBEC3)-driven mutation bias is a key factor in MPXV\u2019s adaptive evolution during its global spread. However, how these mutations affect the structure and function of EFC proteins remains poorly understood. To address this, we performed genomic mutation analysis on globally circulating MPXV clades Ib and IIb, combined with protein monomer, binary, and quaternary complex structure modeling based on AlphaFold 3 and experimental validation by ELISA. We first delineated the mutational spectra of all 11 EFC proteins, revealing that although EFC proteins in clade Ib are highly conserved, lineage IIb B exhibits extensive APOBEC3-driven mutations and the G9 M142I mutation is identified as a lineage-associated APOBEC3-type mutation of lineage IIb B. Structural predictions revealed that while the M142I mutation does not alter G9 monomer folding, it induces a conformational shift in the G9\/A16 subcomplex. Furthermore, within the predicted G9\/A16\/A56\/K2 quaternary complex, this mutation enlarges the interfacial gap and reduces docking stability between the G9\/A16 subcomplex and A56\/K2. Experimental validation demonstrated that the M142I mutation significantly reduces the binding affinity of G9 for A16 and impairs the recruitment of A56\/K2 to the quaternary complex, confirming the computationally predicted mechanism of interface destabilization. These findings highlight a dynamic interplay between APOBEC3-driven evolution and EFC protein structure, demonstrating that the M142I mutation alters EFC complex assembly dynamics and may shift the regulatory balance of the membrane fusion system. These structural changes provide molecular insights into MPXV lineage differentiation, though direct functional assays are required to determine the net effect on viral entry efficiency.<\/jats:p>","DOI":"10.3390\/molecules31091466","type":"journal-article","created":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T15:09:33Z","timestamp":1777388973000},"page":"1466","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Structural Insights into the Impact of the M142I Mutation in Monkeypox Virus G9 Protein on Subcomplex Formation Revealed by AlphaFold 3 Modeling"],"prefix":"10.3390","volume":"31","author":[{"given":"Xudong","family":"She","sequence":"first","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuan","family":"Liang","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linqing","family":"Wang","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifan","family":"Lin","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuenan","family":"Zhang","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Zhu","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qinghua","family":"Wu","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiwei","family":"Xiao","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengsong","family":"Wan","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kexin","family":"Xi","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Zhao","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenguang","family":"Shen","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bao","family":"Zhang","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2986-8028","authenticated-orcid":false,"given":"Jianhai","family":"Yu","sequence":"additional","affiliation":[{"name":"BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, Key Laboratory of Infectious Diseases Research in South China, School of Public Health, Southern Medical University, No. 1023, South Shatai Road, Baiyun District, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1001\/jama.2022.12513","article-title":"The WHO Declaration of Monkeypox as a Global Public Health Emergency","volume":"328","author":"Nuzzo","year":"2022","journal-title":"JAMA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"666","DOI":"10.1056\/NEJMoa2411368","article-title":"Evolving Epidemiology of Mpox in Africa in 2024","volume":"392","author":"Ndembi","year":"2025","journal-title":"N. Engl. J. Med."},{"key":"ref_3","unstructured":"World Health Organization (2025, May 31). Global Mpox Trends. Available online: https:\/\/worldhealthorg.shinyapps.io\/mpx_global\/."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/S1473-3099(23)00055-5","article-title":"New nomenclature for mpox (monkeypox) and monkeypox virus clades","volume":"23","author":"Ulaeto","year":"2023","journal-title":"Lancet Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"102402","DOI":"10.1016\/j.tmaid.2022.102402","article-title":"Phylogenomic analysis of the monkeypox virus (MPXV) 2022 outbreak: Emergence of a novel viral lineage?","volume":"49","author":"Luna","year":"2022","journal-title":"Travel Med. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/j.semcdb.2016.07.015","article-title":"Membrane fusion during poxvirus entry","volume":"60","author":"Moss","year":"2016","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"688","DOI":"10.3390\/v4050688","article-title":"Poxvirus Cell Entry: How Many Proteins Does It Take?","volume":"4","author":"Moss","year":"2012","journal-title":"Viruses"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1636","DOI":"10.1038\/s41564-019-0488-4","article-title":"Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion","volume":"4","author":"Gray","year":"2019","journal-title":"Nat. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Laliberte, J.P., Weisberg, A.S., and Moss, B. (2011). The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components. PLoS Pathog., 7.","DOI":"10.1371\/journal.ppat.1002446"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e2160661","DOI":"10.1080\/22221751.2022.2160661","article-title":"Crystal structure of vaccinia virus G3\/L5 sub-complex reveals a novel fold with extended inter-molecule interactions conserved among orthopoxviruses","volume":"12","author":"Lin","year":"2023","journal-title":"Emerg. Microbes Infect."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2179351","DOI":"10.1080\/22221751.2023.2179351","article-title":"Structural basis of poxvirus A16\/G9 binding for sub-complex formation","volume":"12","author":"Yang","year":"2023","journal-title":"Emerg. Microbes Infect."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Kao, C.F., Tsai, M.H., Carillo, K.J., Tzou, D.L., and Chang, W. (2023). Structural and functional analysis of vaccinia viral fusion complex component protein A28 through NMR and molecular dynamic simulations. PLoS Pathog., 19.","DOI":"10.1101\/2023.06.22.546044"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1569","DOI":"10.1038\/s41591-022-01907-y","article-title":"Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus","volume":"28","author":"Isidro","year":"2022","journal-title":"Nat. Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"8241","DOI":"10.1038\/s41467-023-44092-3","article-title":"Phylogeny and molecular evolution of the first local monkeypox virus cluster in Guangdong Province, China","volume":"14","author":"Yu","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1126\/science.adg8116","article-title":"APOBEC3 deaminase editing in mpox virus as evidence for sustained human transmission since at least 2016","volume":"382","author":"Neher","year":"2023","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1038\/s43856-025-01203-z","article-title":"First imported cases of MPXV Clade Ib from Goma, Democratic Republic of the Congo","volume":"5","author":"Merritt","year":"2025","journal-title":"Commun. Med."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Irfan Khan, M., Saleh, A.A., Ali, R., Jan, R.U., Gu, J., and Dejun, J. (2026). The evolving mpox threat (2022\u20132024): Clade dynamics, immune evasion, and escalating global health challenges. Front. Cell. Infect. Microbiol., 15.","DOI":"10.3389\/fcimb.2025.1677762"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"7452","DOI":"10.1038\/s41467-024-51737-4","article-title":"Evolutionary trajectory and characteristics of Mpox virus in 2023 based on a large-scale genomic surveillance in Shenzhen, China","volume":"15","author":"Zhang","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1016\/j.tips.2023.08.003","article-title":"Differences in pathogenicity among the mpox virus clades: Impact on drug discovery and vaccine development","volume":"44","author":"Andrei","year":"2023","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00093-20","DOI":"10.1128\/JVI.00093-20","article-title":"Experimental Evolution To Isolate Vaccinia Virus Adaptive G9 Mutants That Overcome Membrane Fusion Inhibition via the Vaccinia Virus A56\/K2 Protein Complex","volume":"94","author":"Hong","year":"2020","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e00077-20","DOI":"10.1128\/JVI.00077-20","article-title":"Mutations Near the N Terminus of Vaccinia Virus G9 Protein Overcome Restrictions on Cell Entry and Syncytium Formation Imposed by the A56\/K2 Fusion Regulatory Complex","volume":"94","author":"Cotter","year":"2020","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1516","DOI":"10.3201\/eid3108.241551","article-title":"Emergence of Clade Ib Monkeypox Virus-Current State of Evidence","volume":"31","author":"Satheshkumar","year":"2025","journal-title":"Emerg. Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"9455","DOI":"10.1128\/JVI.01149-06","article-title":"Vaccinia virus F9 virion membrane protein is required for entry but not virus assembly, in contrast to the related L1 protein","volume":"80","author":"Brown","year":"2006","journal-title":"J. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"eaec0254","DOI":"10.1126\/sciadv.aec0254","article-title":"Cryo-EM structure of the vaccinia virus entry fusion complex reveals a multicomponent fusion machinery","volume":"12","author":"Lin","year":"2026","journal-title":"Sci. Adv."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e0072325","DOI":"10.1128\/jvi.00723-25","article-title":"Structural dissection of vaccinia G9 identifies residues essential for membrane fusion and complex assembly","volume":"99","author":"Chiu","year":"2025","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"6266","DOI":"10.1016\/j.cell.2025.07.040","article-title":"Structural basis of poxvirus fusion regulation and anti-A16\/G9 antibody-mediated neutralization and protection","volume":"188","author":"Meola","year":"2025","journal-title":"Cell"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/9\/1466\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T04:16:07Z","timestamp":1778127367000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/9\/1466"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,28]]},"references-count":26,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["molecules31091466"],"URL":"https:\/\/doi.org\/10.3390\/molecules31091466","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,28]]}}}