Phef vsv g

HIV-1 particles were obtained from HEK 293 cells, cotransfected by a combination of three vectors: packaging psPAX2 vector encoding HIV Gag, Pol, Tat and Rev, reporter/transfer pWPXLd-GFP vector encoding LTR, RRE and GFP as a reporter, and envelope pHEF-VSV-G vector, encoding vesicular stomatitis virus Env, VSV-G. The psPAX2 vector [32 (link)] was kindly provided by Dr. Luban, the pWPXLd-GFP and pHEF-VSV-G vectors were purchased from Addgene (Watertown, MA, USA).
HEK-293 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (Sigma) and 1% L-glutamine (Sigma) at 37 °C under 5% CO₂. A day before transfection, cells were plated at 3 × 105 cells per well. The following day, cells were transfected with the appropriate vectors using polyethylenimine (PEI, 1 mg/mL) at a 2:1 PEI:DNA ratio. Four hours post-transfection, the culture medium was replaced with fresh DMEM, containing various concentrations of tested compounds, solubilized in DMSO. At 48 h post-transfection, the culture media containing released virions were harvested, filtered through 0.45-µm pores membrane and used for immunochemical quantification and characterization by ELISA and Western blot using rabbit anti-HIV-1 CA antibody.

HIV-1 particles were obtained from HEK 293 cells, cotransfected by a combination of three vectors: packaging psPAX2 vector encoding HIV Gag, Pol, Tat, and Rev; reporter/transfer pWPXLd-GFP vector encoding LTR, RRE, and GFP as a reporter; and envelope pHEF-VSV-G vector, encoding vesicular stomatitis virus Env, VSV-G. The psPAX2 vector was kindly provided by Dr. Jeremy Luban; the pWPXLd-GFP and pHEF-VSV-G vectors were purchased from Addgene. HEK-293 cells were grown in Dulbecco’s modified Eagle medium (DMEM, Sigma, St. Louis, MI, USA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MI, USA) and 1% l-glutamine (Sigma, St. Louis, MI, USA) at 37 °C under 5% CO₂. A day before transfection, cells were plated at 3 × 10⁵ cells per well. The following day, cells were transfected with the appropriate vectors using polyethylenimine (PEI, 1 mg/mL) at a 2:1 PEI:DNA ratio. Four hours post transfection, the culture medium was replaced with fresh DMEM, containing various concentrations of tested compounds, solubilized in DMSO. At 48 h post-transfection, the culture media containing released virions were harvested, filtered through 0.45-µm pores membrane, and used for immunochemical quantification and characterization by Western blot using rabbit anti-HIV-1 CA antibody.

HeLa (ARP 154) cells obtained from the NIH AIDS reagent program and LentiX293T cells (Clonetech Laboratories, Mountain View, CA, USA; #632180) were grown in Dulbecco modified Eagle’s Medium (DMEM) supplemented with 10% FetalClone III Serum (HyClone Laboratories, Logan, UT, USA; #SH30109.03). Lentivirus was produced from transfected LentiX293T cells. Briefly, 6 well plates were seeded with 0.6 million cells and incubated for 24 h prior to transfection. Cells were co-transfected with gXRCC4-lenticrisprV2/gScrambled-lenticrisprV2, 1.2 µg; psPAX-2 (Addgene Watertown, MA, USA; #12260), 1.0 μg, and pHEF-VSVG (Addgene #22501), 0.3 μg using the Lipofectamine LTX transfection reagent (Invitrogen, Waltham, MA, USA) at a 1:3 ratio [DNA (μg): Transfection reagent (μL)]. After 6 h of incubation, media was replaced, and cells were cultured in complete media for 48 h. Cell supernatant were collected, filtered through a 0.45 μm syringe filter (MilliporeSigma, Burlington, MA, USA), and used for transduction into HeLa cells.