Fig. 1: The VACV protein C6 induces proteasomal degradation of TRIM5α.

a, Temporal abundance of TRIM5 during VACV-WR infection of TERT-immortalized human fetal foreskin fibroblast (HFFF-TERT) cells measured by mass spectrometry. n = 3 per condition (from ref. ¹⁰). Cytosine arabinoside (AraC) was added where indicated. b–f, Immunoblots showing TRIM5α abundance in: VACV-infected HeLa cells at the indicated times post-infection with (+) or without (−) MG132 (b); HeLa cells after infection with VACV or mutant v6/2 lacking genes near the left genomic terminus (c); HFFF-TERT cells after infection with VACV-WR WT or mutant v∆C6 lacking the gene C6L (d); HEK293T cells inducibly expressing Myc-tagged C6 (+150 ng ml⁻¹ doxycycline (Dox) for 24 h) (e); and T-REx-293 TRIM5^−/− cells complemented with inducible expression of the TAP-tagged TRIM5α mutant N70A and infected with WT VACV-WR or vΔC6 (f). g, Endogenous C6 and TRIM5α co-precipitate during VACV infection. HEK293T cells were infected with VACV-expressing TAP-tagged C6 or N1. HDAC5 was used as a positive control for C6 co-precipitation (unpublished data). h, Mapping of the TRIM5α domains required for C6 interaction. T-REx-293 TRIM5^−/− cells were co-transfected with HA-tagged C6 or N1 and TAP-tagged TRIM5α WT and mutants lacking domains, which are indicated in the right schematics. In b,d,f, MG132 was added at 2 h post-infection (hpi) (+). In g,h, TAP-tagged proteins were precipitated using Strep-Tactin beads. In b–h, inputs and affinity-purified (AP) proteins or protein extracts were analysed by SDS–PAGE and immunoblotted for the indicated epitope or protein. GAPDH, α-actin and D8 were controls for equal loading and viral infection, respectively. Data from b–h are representative of three independent experiments.