Generation of HIV-1 Reporter Virus Stocks

Replication-competent reporter virus stocks were generated from an HIV-1 NL4-3 molecular clone in which GFP had been cloned behind an IRES cassette following the viral nef gene (NIH AIDS Reagent Program, catalog no. 11349). Briefly, 10 mg of the molecular clone was transfected (PolyJet; SignaGen) into 5 × 10⁶ human embryonic kidney (HEK) 293T cells (ATCC, CRL-3216) according to the manufacturer’s protocol. Twenty-five milliliters of the supernatant was collected at 48 and 72 hours and then combined. The virus-containing supernatant was filtered through 0.45-mm polyvinylidene difluoride filters (Millipore) and precipitated in 8.5% polyethylene glycol [average molecular weight (Mn), 6000; Sigma-Aldrich] and 0.3 M NaCl for 4 hours at 4°C. Supernatants were centrifuged at 3500 rpm for 20 min, and the virus was resuspended in 0.5 ml of PBS for a 100× effective concentration. Aliquots were stored at −80°C until use.