ORF3a is a key driver of maternal SARS-CoV-2 infection-associated placental dysfunction

SARS-CoV-2 infection during pregnancy is associated with an increased risk of pre-eclampsia (PE), a hypertensive disorder, but the molecular mechanisms remain poorly understood. Here, we identify ORF3a, a SARS-CoV-2 accessory protein, as a key factor in placental dysfunction, driving autophagy dysregulation, trophoblast maturation impairment, protein aggregation and placental barrier disruption– processes linked to PE. We detect ORF3a in placentas from women infected with SARS-CoV-2 along with increased protein aggregation and disrupted tight junctions in ORF3a + regions. In placental cell lines, ORF3a impairs syncytiotrophoblast maturation and induces protein aggregation. Mechanistically, ORF3a binds to ZO-1 via its PDZ-binding motif (SVPL), and deletion of this domain from ORF3a abrogates its effect on trophoblast barrier integrity. In human trophoblast cells engineered with an LC3-GFP-mCherry reporter, ORF3a induces autophagosome accumulation, and shifts autophagy toward a secretory pathway with elevated levels of CD63 + extracellular vesicles and disrupted ZO-1 localization, all of which are recapitulated by live infection with the SARS-CoV-2 Delta variant. These ORF3a-dependent changes are fully recapitulated in 3D stem-cell-derived trophoblast organoids (SC-TOs). Together, our findings define a molecular mechanism by which SARS-CoV-2 infection compromises placental syncytial integrity. Targeting ORF3a may provide a therapeutic strategy to mitigate PE-like placental dysfunction in SARS-CoV-2–infected pregnancies.