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.