Fig. 5: Conservation of C6 and L3 in orthopoxviruses and the effect of CsA and derivatives.
From: TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6
a, Immunoblot showing TRIM5α in T-REx-293 cells inducibly expressing TAP–C6 proteins from VACV-WR, RPXV, CPXV, elephantpox virus, CMLV, MPXV UK2022, MPXV Zaire and VARV India 1967. The numbers indicate the band intensity of TRIM5α normalized to α-actin and relative to non-induced control. b, Immunoblot showing TRIM5α in HEK293T cells infected with CPXV or CMLV, and HeLa cells infected with MPXV-CVR-S1. Cells were infected at 5 PFU per cell. MG132 was added at 2 hpi. c, MPXV UK2022, MPXV Zaire, VARV and VACV L3 co-precipitation with TRIM5α and CypA. T-REx-293 TRIM5−/−CypA−/− cells were transfected with HA–L3 and TAP–TRIM5α, ΔSPRY or CypA ± CsA 24 h before harvest. d, Effect of CsA and derivatives on infectious VACV titre following infection in T-REx-293 cells at 5 PFU per cell for 16 h. n = 3 per condition. e, Effect of CsA and derivatives on infectious MPXV-CVR-S1 titres following infection in HFFF-TERT cells at 0.01 PFU per cell for 72 h. n = 3 per condition. f, Effect of CsA and derivatives on infectious MPXV-CVR-S1 titres following infection in HFFF-TERT cells at 5 PFU per cell for 18 h. n = 3 per condition. In a, cells were treated with 150 ng ml−1 doxycycline for 16 h before harvest. Protein extracts (b) and inputs and AP proteins (c) were analysed by immunoblotting. In d–f, DMSO, CsA, NIM811 or alisporivir were added at 1 hpi. Data from c are representative of three independent experiments, whereas a,b,d–f are from two independent experiments. Data from d–f were analysed using one-way Welch’s ANOVA test (where P < 0.0001 for d), and pairwise comparisons in d were performed using post-hoc Dunnett’s T3 multiple comparisons test. Analyses were performed on GraphPad Prism. Data are mean ± s.e.m. (d–f).
