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We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins, which potentially mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for those ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Rab geranyl geranylation emerged as a significant hit, and also Rab proteins, which are crucial regulators of the endocytic pathway and interactors of both p34 and E199L. Rab proteins co-ordinate a tight regulation of the endocytic pathway that is necessary for ASFV infection. Moreover, several interactors were proteins involved in the molecular exchange at ER membrane contacts. These ASFV fusion proteins shared interacting partners, suggesting potential common functions. Membrane trafficking and lipid metabolism were important categories, as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages.<\/jats:p>","DOI":"10.3390\/v15051098","type":"journal-article","created":{"date-parts":[[2023,5,1]],"date-time":"2023-05-01T12:02:31Z","timestamp":1682942551000},"page":"1098","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Elucidation of the Cellular Interactome of African Swine Fever Virus Fusion Proteins and Identification of Potential Therapeutic Targets"],"prefix":"10.3390","volume":"15","author":[{"given":"Isabel","family":"Garc\u00eda-Dorival","sequence":"first","affiliation":[{"name":"Departmento de Biotecnolog\u00eda, INIA-CSIC, Centro Nacional Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de la Coru\u00f1a Km 7.5, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miguel \u00c1ngel","family":"Cuesta-Geijo","sequence":"additional","affiliation":[{"name":"Departmento de Biotecnolog\u00eda, INIA-CSIC, Centro Nacional Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de la Coru\u00f1a Km 7.5, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8257-9797","authenticated-orcid":false,"given":"Inmaculada","family":"Galindo","sequence":"additional","affiliation":[{"name":"Departmento de Biotecnolog\u00eda, INIA-CSIC, Centro Nacional Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de la Coru\u00f1a Km 7.5, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana","family":"del Puerto","sequence":"additional","affiliation":[{"name":"Departmento de Biotecnolog\u00eda, INIA-CSIC, Centro Nacional Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de la Coru\u00f1a Km 7.5, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luc\u00eda","family":"Barrado-Gil","sequence":"additional","affiliation":[{"name":"Departmento de Biotecnolog\u00eda, INIA-CSIC, Centro Nacional Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de la Coru\u00f1a Km 7.5, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7870-4580","authenticated-orcid":false,"given":"Jes\u00fas","family":"Urquiza","sequence":"additional","affiliation":[{"name":"Departmento de Biotecnolog\u00eda, INIA-CSIC, Centro Nacional Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de la Coru\u00f1a Km 7.5, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0862-6177","authenticated-orcid":false,"given":"Covadonga","family":"Alonso","sequence":"additional","affiliation":[{"name":"Departmento de Biotecnolog\u00eda, INIA-CSIC, Centro Nacional Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaria, Ctra. de la Coru\u00f1a Km 7.5, 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3018","DOI":"10.1111\/tbed.14341","article-title":"African swine fever in the Dominican Republic","volume":"68","author":"Gonzales","year":"2021","journal-title":"Transbound. 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