Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

Background: Viruses initiate infection by engaging specific receptors on the host cell surface. While the surface receptor ACE2 mediates SARS-CoV-2 entry, the precise role of subcellular trafficking, particularly, the exact involvement of primary cilium trafficking in viral entry remains largely unresolved. Methods: We used in-vitro human cell models and SARS-CoV-2 pseudoviruses to elucidate the viral attachment and host cell entry mechanisms. Mechanistic studies were conducted using a multidimensional approach that combined flow cytometry analysis, co-immunofluorescence and confocal microscopy, co-immunoprecipitation, genetic manipulations and ciliary perturbations, and structural predictions. Results: Our study uncovers Tubby Like Protein-3 (TULP3) as a pivotal ciliary trafficking adaptor that facilitates ACE2 localization to the primary cilium. We show that ACE2 and TULP3 physically associate, and that TULP3 depletion not only removes ACE2 from the ciliary axoneme but also impairs SARS-CoV-2 pseudovirus entry. This ACE2 localization is partially dependent on TULP3's interaction with the IFT-A complex, as an IFT-A-binding-deficient TULP3 mutant could still partially rescue ciliary ACE2 levels. Furthermore, genetic disruption of ACE2-enriched primary cilia in human lung cells and retinal pigment epithelial cells significantly diminishes the infectivity of SARS-CoV-2 pseudoviruses, including the ancestral, Delta and Omicron BA.5 variants. Viral spike protein attachment assays reveal preferential binding and accumulation of the SARS-CoV-2 spike on ACE2-rich ciliary axonemes. Moreover, we demonstrate variable endogenous enrichment of ACE2 within the primary cilium axoneme across diverse SARS-CoV-2 susceptible human cell types, including lung epithelial cells, retinal pigmented epithelial cells, neuroblastoma cells, and human iPSC-derived neural progenitors, offering a potential mechanistic framework for tissue-specific susceptibility and the heterogeneous clinical manifestations of COVID-19. Conclusion: Our findings demonstrate the first evidence of a dedicated ciliary trafficking machinery for ACE2. We provide compelling evidence that SARS-CoV-2 hijacks evolutionarily conserved ciliary trafficking pathways, with TULP3-dependent targeting of ACE2 to primary cilia serving as a determinant of viral host cell tropism and invasion. This work uncovers novel molecular mechanisms underpinning SARS-CoV-2 infection, and highlights the primary cilium as a critical nexus for viral entry.