Pseudotyped HIV and MLV Particle Production

Replication-incompetent HIV particles pseudotyped with VSV-G were generated by calcium phosphate transfection of HEK-293T cells (ATCC) with 20 μg of proviral HIV vector and 12 μg of pL-VSV-G plasmid (36 (link)) per 3 × 10⁶ cells seeded on 10-cm plates. The transfection media was replaced after 8 h with fresh DMEM supplemented with 10% FCS. Supernatants were collected at 48 h after transfection. The virus-containing supernatants were centrifuged for 10 min at 1,200 rpm to remove cells, then passed through 0.4-μm filters to remove fine debris. Supernatants either were used immediately for infections or were frozen in aliquots at −80°C. The viral titers were determined by infection of the human T cell line Hut78 with serially diluted virus supernatant. Typically, viral titers had a range of 3–10 × 10⁶ infectious units (ifu)/ml. T cells were infected at a multiplicity of infection (MOI) of 10–20 in 24-well plates in the presence of 10 μg/ml polybrene (Sigma Chemical Co.). After an additional day of culture with virus supernatants, cells were washed or sedimented through Ficoll and resuspended in fresh culture media. R5-tropic replication-competent viruses were prepared similarly by transfecting 293T cells, and titers of 2–5 × 10⁵ ifu/ml were obtained. VSV-G–pseudotyped MLV-based viruses were similarly prepared by transfecting 293T cells with 12 μg each of pMX.EGFP, pJK3 (expressing MLV gag and pol genes), and pL-VSV-G as well as 3 μg of pCMV-Tat plasmids.