{"doi":"10.1101/2024.09.25.614931","title":"SARS-CoV-2 ORF3a Protein Impairs Syncytiotrophoblast Maturation, Alters ZO-1 Localization, and Shifts Autophagic Pathways in Trophoblast Cells and 3D Organoids","abstract":"SARS-CoV-2 infection poses a significant risk to placental physiology, but its impact on placental homeostasis is not well understood. We and others have previously shown that SARS-CoV-2 can colonize maternal and fetal placental cells, yet the specific mechanisms remain unclear. In this study, we investigate ORF3a, a key accessory protein of SARS-CoV-2 that exhibits continuous mutations. Our findings reveal that ORF3a is present in placental tissue from pregnant women infected with SARS-CoV-2 and disrupts autophagic flux in placental cell lines and 3D stem-cell-derived trophoblast organoids (SCTOs), impairing syncytiotrophoblast differentiation and trophoblast invasion. This disruption leads to protein aggregation in cytotrophoblasts (CTB) and activates secretory autophagy, increasing CD63+ extracellular vesicle secretion, along with ORF3a itself. ORF3a also compromises CTB barrier integrity by disrupting tight junctions via interaction with ZO-1, mediated by its PDZ-binding motif, SVPL. Colocalization of ORF3a and ZO-1 in SARS-CoV-2-infected human placental tissue supports our in vitro findings. Deleting the PDZ binding motif in the ORF3a protein (ORF3a-noPBM mutant) restored proper ZO-1 localization at the cell junctions in an autophagy-independent manner. Lastly, we demonstrate that constitutive ORF3a expression induces SC-TOs to transition towards a secretory autophagy pathway likely via the PBM motif, as the ORF3a-NoPBM mutants showed a significant lack of CD63 expression. This study demonstrates the functional impact of ORF3a on placental autophagy and reveals a new mechanism for the activation of secretory autophagy, which may lead to increased extracellular vesicle secretion. These findings provide a foundation for exploring therapeutic approaches targeting ORF3a, specifically focusing on its PBM region to block its interactions with host cellular proteins and limiting placental impact.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":491961,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9514,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":460947,"name":"Rowan M. Karvas","orcid":"0000-0003-0188-8737","position":1,"is_corresponding":false},{"id":998519,"name":"Brittany L. Jones","orcid":"0000-0002-2715-593X","position":2,"is_corresponding":false},{"id":1338271,"name":"Eliza McColl","orcid":null,"position":3,"is_corresponding":false},{"id":1338272,"name":"Emily Diveley","orcid":null,"position":4,"is_corresponding":false},{"id":685553,"name":"Sukanta Jash","orcid":null,"position":5,"is_corresponding":false},{"id":328586,"name":"Surendra Sharma","orcid":"0000-0002-4331-3467","position":6,"is_corresponding":false},{"id":649969,"name":"Jeannie C. Kelly","orcid":"0000-0002-4908-4693","position":7,"is_corresponding":false},{"id":233616,"name":"Thorold W. Theunissen","orcid":"0000-0001-6943-7858","position":8,"is_corresponding":false},{"id":314987,"name":"Indira U. Mysorekar","orcid":"0000-0003-3917-8677","position":9,"is_corresponding":false},{"id":844348,"name":"Deepak Kumar","orcid":"0000-0001-8182-0016","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:08:49.768795Z","pmid":"39386577","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}