Lentivirus Production and Chlamydia Infection

For lentivirus production, 293FT cells were transfected with corresponding vectors using FuGene HD transfection (Promega, USA), following the manufacturer’s instructions. Packaging vectors were psPAX.2 and psMD2.G (Addgene Plasmids, #12260 and #12259; Dr Didier Trono). Virus-containing supernatant was collected, filtered and incubated with HeLa cells in the presence of 1 μg/ml of polybrene (Millipore, #TR-1003-G).
Chlamydia trachomatis serovar L2 (Ctr) was obtained from the American Type Culture Collection and propagated in HeLa cells. Ctr strains deficient in expression of chlamydial protease/proteasome-like activity factor (CPAF) (Rst17) and the isogenic, CPAF competent strain (Rst5) [35 (link)] were a kind gift from Dr Raphael Valdivia (Duke University School of Medicine, Durham, North Carolina, USA). Bacteria were purified over a Gastrografin density gradient (Bayer Vital, Leverkusen), followed by titration on HeLa cells and stored in SPG medium (0.2 M sucrose, 8.6 mM Na2HPO4, 3.8 mM KH2PO4, 5 mM glutamic acid [pH 7.4]) at −80 °C. Fresh aliquots were thawed for each experiment. Cells were infected at a multiplicity of infection (MOI) of 5 in complete culture medium.