Fig. 3

A1 and A2 Exogenous administration of α-syn monomers increases SARS-CoV-2 replication in A549-hACE2 and CaLu-3 epithelial cells, which is prevented by IFN-β. Quantification of viral replication from cell supernatants through RT-qPCR for the SARS-CoV-2 N gene in infected A549-hACE2 (A1) and CaLu-3 cells (A2), 24 and 48 h post-infection (MOI 0.05). A1 In A549-hACE2 cells, exogenous α-syn monomers significantly increased viral replication compared with infected CTR both at 24 and 48 h post-infection. A2 In CaLu-3 cells the pro-viral effect of exogenous α-syn monomers was maximally detected at 24 h, while it declined at 48 h post-infection. In both cases, IFN-β, either alone or in combination with exogenous α-syn, completely prevented SARS-CoV-2 replication. Results show SARS-CoV-2 N gene copy numbers quantified from the RNA isolated from 100 μl of cell supernatants. Results are shown as mean ± SEM from n ≥ 5 independent experiments. Values for 24 and 48 h post-infection were analyzed by applying One-Way ANOVA followed by multiple testing correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B1 and B2 Representative immunofluorescence images of SARS-CoV-2 Nucleocapsid (N) and α-syn proteins in Mock- and SARS-CoV-2-Infected A549-hACE2 epithelial lung cells 48 h post-infection (MOI 0.05), in the absence and presence of IFN-β, exogenous α-syn monomers, or both. SARS-CoV-2 replication is enhanced in the presence of exogenous α-syn monomers, which impair accumulation of permeabilization-resistant α-syn species that are instead increased by IFN-β. B1 For visualization of large, permeabilization-resistant α-syn species, that potentially correspond to multimers or intracellular membrane-bound species, cells were fixed for 15 min in 4% Formaldehyde solution and permeabilized with 0.3% of Triton X-100 for 15 min. Bars correspond to 20 μm. B2 For better preservation, and visualization of small, highly-soluble α-syn species potentially corresponding to monomers, cells were fixed for 15 min in 4% Paraformaldehyde (PFA) and permeabilized with 0.1% of Triton X-100 for 10 min. Bars correspond to 20 μm. Images are representative of n = 3 independent experiments. C Quantification of SARS-CoV-2-N-positive cells in SARS-CoV-2-infected CTR vs α-syn-treated SARS-CoV-2-infected cells. Values are expressed as the percentage of N-positive cells ± SD calculated as the number of N-positive cells/total cells per microscopic field from n = 12 microscopic fields per experimental group, from n = 3 independent experiments. Data were analyzed by applying the Student’s t-test for comparison between VIRUS CTR and VIRUS α-syn groups. **p < 0.01