Lentiviral Vector Production and Cell Depletion

293T CRL-3216 cells were purchased from ATCC and authenticated by the supplier. All cells are regularly tested and are mycoplasma free. Gag/Pol and GFP vectors were, respectively, for HIV-1 pCRV-1(Zennou et al., 2004 (link)) and CSGW(Naldini et al., 1996 (link)); for HIV-2, HIV-2 ROD Gag/Pol and HIV-2 GFP(Griffin et al., 2001 (link)); for HIV-1 O p8.91 MVP(Ikeda et al., 2004 (link)); for SIVmac, SIV3⁺ and SIV-eGFP(Poeschla et al., 1998 (link)) and for FIV FP93(Poeschla et al., 1998 (link)). Lentiviral packaging plasmid pMDG2, which encodes VSV-G envelope, was used to pseudotype infectious virions (Addgene plasmid # 12259). Cells for depletion of TNPO3 and NUP358 were produced by expression of shRNAs as previously described(Schaller et al., 2011 (link)). HEK293T and HeLa cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO) at 37°C with 5% CO₂. PBMCs were stimulated with 2 μg/ml PHA-P for 4 days before infection, then cultured in RPMI 1640 with 10% FBS, 2mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 U/ml IL-2.