Fig 2: Scheme depicting features of SARS-CoV-2 N protein during infection. N protein undergoes proteolysis to produce N1–209, N1–220, N1–273, N156–419, and N1–392. NFL and N proteoforms bind RNA with a preference for structured RNA, and NFL dimers are likely functional unit of assembly in ribonucleoprotein complexes. Immunophilin CypA binds directly to N1–209 but not NFL, and the interaction can be inhibited through addition of cyclosporin A (CsA). N156–419 and NFL interact with antibodies from convalescent plasma, while proteoforms N1–209, N1–220, and N1–273 fail to induce the same antibody response.

Fig 3: N proteoforms directly interact with cyclophilin A. (A) Mass spectrum of N1–209 after incubation with cyclophilin A (CypA) in a 1:1 molar ratio. The mass spectrum at m/z > 3000 was magnified 5× and offset for clarity (red trace). Three charge state distributions that correspond to a low population of homodimers of N1–209, homodimers of CypA, and heterodimers of N1–209–CypA. (B) Mass spectrum of N1–209 after incubation with CypA and cyclosporin A (CsA) in a molar ratio of 1:1:2. CypA preferentially binds CsA as demonstrated by the charge state distribution centered at 8+, which corresponds to the CypA–CsA complex. The mass spectrum at m/z > 3500 was magnified 5× and offset (red trace) to highlight the exceedingly low abundance of N1–209–CypA heterodimers that persist after CsA treatment. The scheme (inset of B) depicts CsA competitively binding to CypA and abolishing the N1–209–CypA interaction.

Fig 4: Effect of compounds 1, 2, and GraL over human T lymphocytes migration. Cells were pre-treated with compound 1, 2 (0.1 µM), GraL (1 μM) or CsA (0.2 µM) for 2 h and then were stimulated with Con A (50 μg/mL) for 48 h. Then, T cells were placed in the migration chamber for 24 hours in the presence of the chemoattractant CypA (50 ng/mL) (A) or CypB (5 ng/mL) (B). Controls of untreated T cells in the presence or absence of Cyps are also included. Data are the result of average ± SEM (n = 3). The values are shown as the difference between Con A migratory cell numbers compared to cells treated with compounds plus Con A, *p < 0.05 and **p < 0.01. Values of cells treated with Con A were compared with cells in the presence of CypA or B, # p < 0.05. Values of untreated cells in absence of CypA or B were compared with untreated cells in presence of CypA and B, + p < 0.05 or ++ p < 0.01. One-way ANOVA test with Dunnet’s post hoc analysis and Student´s t-test were used for statistical analysis.

Fig 5: Effect of compounds 1, 2, and GraL over intra and extracellular CypA, B and C levels in human T lymphocytes stimulated with Con A Cells were pre-treated with compound 1, 2 (0.1 µM), GraL (1 μM) or CsA (0.2 µM) for 2 h and then were stimulated with Con A (50 μg/mL) for 48 h. CypA (A), CypB (B), and CypC (C) levels were measured by ELISA in the medium of T lymphocytes. The line represents the unstimulated cells values. The intracellular expression of CypA (D), CypB (E), and CypC (F) was measured by western blot in cytosolic lysates of T lymphocytes. Band intensity was normalized by β-actin. Data are the result of average ± SEM (n = 3). The values are shown as the difference between cells treated with Con A and cells treated with compounds plus Con A, *p < 0.05, **p < 0.01 and ***p < 0.001. Values of cells treated with Con A were compared with control cells, # p < 0.05, ## p < 0.01 and ### p < 0.001. One-way ANOVA test with Dunnet’s post hoc analysis and Student´s t-test were used for statistical analysis.