Tapi 1

The titers of the virus stocks were determined using TaqMan qRT-PCR for HIV-1 RNA (target in integrase) and normalized to a nominal 400 virion equivalents per cell for infection. Infections were performed by spinoculation in the presence of 5 µg/ml DEAE dextran (Sigma) for 2 h at 1,200×g and 37°C [22] (link), [132] (link). Cells were then washed and plated. For FKHR TransAM experiments, IL-7 treated CD4 T cells were infected with NLENHSA-ESI. At 7 dpi., cells were ficolled in order to remove dead cells, then stained with a phycoerythrin (PE) labeled anti-mCD24 and bound to anti-PE magnetic microbeads (Miltenyi Biotec). Positive and negative cells were separated using the MACS system (Miltenyi Biotec). Purity was routinely >95%.
Cell treatments included PMA (2 nM, Fisher Scientific), LY294002 (5 µM, BioVision), PI-103 (5 µM, EMD Millipore), TAPI-1 (50 µM, Santa Cruz Biotechnology). Non-nucleoside reverse-transcriptase inhibitor Efavirenz (2 µg/ml) and Protease inhibitor Indinavir (2 µM) were obtained from the NIH AIDS Research and Reference Reagent Program. The Foxo1 inhibitor 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856) [78] (link), was purchased from EMD Millipore and used at a final concentration of 50 nM. DMSO controls were performed with a corresponding dilution of 1 in 20,000.

Metalloprotease inhibitors, including BB94 (batimastat), TAPI-1 and TAPI-2 (Santa Cruz Biotechnology), and TIMP1, TIMP2, TIMP3, and TIMP4 (R&D Systems), were added at the time of GPR37L1 induction (or 5 h after transfection in the case of U87 MG cells) or 24 h after induction for TX14A experiments, and cells were then incubated for 24 h before lysis. For MG132 proteasome inhibition, cells were induced with doxycycline overnight and then treated with MG132 for 6 h before harvest. Verification of TIMP activity in vitro using activated MMP-2 and the fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ was performed as per the protocol provided by R&D Systems.