Quantitative PCR analysis of NF-κB genes

Quantitative PCR with reverse transcription (RT–qPCR) analysis of NF-κB-responsive genes was undertaken as previously described^{61 (link)}. In brief, the TRIM5^−/−CypA^−/− cell line was modified to express inducibly either VACV TAP-tagged-L3 or empty vector. These cells were seeded in 12-well plates at a density of 6 × 10⁵ cells per well; the next day, these cells were incubated in medium without serum for 3 h and then either mock-treated or treated with doxycycline (150 ng ml⁻¹) for 4 h in triplicate. Total RNA was extracted from the cells and cDNA was synthesized by reverse transcription using oligo-dT primers (Thermo Fisher). The mRNA levels of the NF-κB-responsive genes NFKBIA, CCL2, CXCL8, CXCL10, IL6 and the housekeeping control gene GAPDH were measured by qPCR using Fast SYBR Green master mix (Thermo Fisher) and a ViiA 7 real-time PCR machine (Thermo Fisher). The fold induction of the mRNA levels was calculated by the 2^−ΔΔCt method using induced T-REx-293 empty vector and GAPDH as the internal control. The primers used in the qPCR are listed in Supplementary Table 2.