Extended Data Fig. 8: Conservation of L3 in VARV and MPXV and the effect of CsA and derivatives.
From: TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6
a) Amino acid sequence alignment of L3 proteins from orthopoxviruses created as in Extended Data Fig. 7a. b) Effect of CsA and derivatives, NIM811 or alisporivir, on MPXV UK2022 L3 co-precipitation with CypA. T-REx-293 TRIM5-/-CypA-/- cells were co-transfected with HA-tagged L3 and TAP-tagged TRIM5α, ΔSPRY or CypA +/− 20 μM CsA, NIM811 or alisporivir 20 h before harvest. Graph shows band intensity of HA-tagged MPXV L3 in the rightmost 4 lanes from three independent experiments, normalised to TAP-tagged CypA and relative to the DMSO carrier sample. c) Effect of CsA and derivatives, NIM811 and alisporivir on endogenous VACV L3 co-precipitation with CypA. T-REx-293 TRIM5-/-CypA-/- cells were co-transfected with TAP-tagged TRIM5α, ΔSPRY or CypA and infected with vΔC6 at 3 PFU/cell +/− 10 μM CsA, NIM811 or alisporivir for 12 h. The graph shows band intensity of VACV L3 from the rightmost 4 lanes from three independent experiments, normalised to TAP-tagged CypA and relative to the DMSO carrier sample. d) Effect of CsA on virus titres following VACV infection in T-REx-293 CypA-/- cells at 5 PFU/cell for 16 h. n = 3/condition. e) Effect of CsA on virus titres following VACV infection in T-REx-293 TRIM5-/- cells at 5 PFU/cell for 16 h. n = 3/condition. f) Plaque area quantification of MPXV-CVR-S1 infection +/− 5 μM CsA and derivatives. BS-C-1 cells were infected with MPXV-CVR-S1 and 5 μM CsA, NIM811 or alisporivir were added to the semi-solid overlay. Plaques were measured using ImageJ and area relative to DMSO carrier were calculated. n ≥ 19/condition. g) Effect of CsA on plaque size following RPXV and CMLV infection in BS-C-1 cells. CsA at the indicated concentrations were added at 1 hpi. Plaques were measured using ImageJ and their area relative to DMSO carrier were calculated. n = 69/condition for CMLV and n = 68/condition for RPXV. Inputs and AP proteins in b) and c) were analysed by SDS-PAGE and immunoblotted for the indicated epitope/protein. Data shown in b) and f) is representative of four independent experiments, c) is from three and d), e) and g) are from two. Data from b), c), d) and g) were analysed using One-Way Welch’s ANOVA test. Data from e) and f) was analysed using two-tailed unpaired Student’s t-test. Analyses were performed on GraphPad Prism. Mean ± s.e.m.
