Padvantage vector

Beta-lactamase-Vpr (BlaM-Vpr)-containing HIV-1 particles were generated to assay HIV-1 fusion, as described by Cavrois et al.^{28 (link)}. The two HIV-1 proviral clones used, which expressed either the X4 Env from HIV-1 NL4-3 or the R5 Env from HIV-1 JR-FL, were a kind gift from Dr. Bernard Lagane (CPTP, Toulouse, France), and have been reported previously^{57 (link)}. These proviral clones, which are derived from pNL4-3, carry a luciferase reporter gene in place of the nef gene, and differ in a fragment encompassing the env gene between positions 6113 and 8797. To produce BlaM-Vpr HIV-1 particles, 8 × 10⁶ HEK 293 T cells in 162 cm² culture flasks were cotransfected using the calcium phosphate-DNA co-precipitation method with 60 μg proviral DNA, 20 μg pCMV-BlaM-Vpr plasmid, and 10 μg pAdVAntage vector (Promega), which increases transient protein expression. The culture media was replaced 18 h post-transfection, and supernatants containing viral particles were harvested at 48 h. The supernatants were clarified at low speed (1460 g) and then ultracentrifuged at 23,000 g for 90 min at 4 °C. The pelleted viruses were resuspended in DMEM 10% FBS at a final 50x concentration, quantified for their content in HIV-1 Gag p24 antigen (Alliance HIV P24 antigen ELISA Kit from PerkinElmer), and stored at −80 °C before use.

The Blam-Vpr based entry assay was applied to study the effect of IFN-γ on viral entry as described previously [36 (link)]. Briefly, 293T cells were cotransfected with pNL4-3. Luc. R^- E^-, pCMV-Blam-Vpr (NIH AIDS Research and Reference Reagent Program), pAdVAntage vector (Promega), and plasmids encoding viral GP protein to produce Blam-Vpr chimera pseudoviral particles. A549 cells were treated with IFN-γ and inoculated with Blam-Vpr pseudoviral particles. CCF2 substrate was loaded into cells at 1 h after infection and then washed three times with PBS. At 24-h postinfection, the cells were fixed with 2% formaldehyde and analysed by flow cytometry.

Transfections were performed in 25-cm² flasks with the cells ~60–70% confluent, using Fugene transfection reagent (Roche),5μg plasmid and 0.5μg of pAdvantage vector (Promega) to increase the translation efficiency of the chondroitinase gene. Transfections were performed according to the manufacturer’s instructions. 24h post-transfection, the medium was replaced with Neu7 conditioned medium as a source of CSPGs. This CSPG-containing medium was incubated with the transfected cells for 24h to allow digestion of the CSPGs by any ChABC produced as a result of the transfections. It was then harvested, centrifuged to remove detached cells, and concentrated 10- fold in a centricon-50 unit (Millipore). Protease inhibitor cocktail (Sigma) was added and the medium frozen for subsequent analysis by western blot. In every round of transfection, transfection efficiency was determined using a plasmid encoding GFP

BlaM-vpr-containing viral clones were prepared in HEK 293T cells as described [110 (link)]. Briefly, 1.5 x 10⁷ cells in 162 cm² culture flasks were cotransfected using the calcium phosphate-DNA coprecipitation method with 60 μg proviral DNA, 20 μg pCMV-BlaM-vpr plasmid and 10 μg pAdvantage vector (Promega). Forty-eight hours post-transfection, culture supernatants containing the viral particles were clarified at low speed and then ultracentrifuged at 72,000 g for 90 min at 4°C. The pelleted viruses were then resuspended in DMEM, measured for their content in HIV-1 Gag p24 antigen (Alliance HIV P24 antigen ELISA Kit from PerkinElmer) and stored at -80 °C before use.
Fifty ng Gag p24 of BlaM-vpr-containing viruses were exposed to 2 x 10⁵ T-cells or 1.5 x 10⁵ MDMs, which were detached from culture flasks with Cellstripper (Corning), as previously described [42 (link)]. Then, cells were incubated for 2 h with the CCF2/AM dye (using the CCF2-AM loading kit from Invitrogen). Loading of the A3.01 T cells with CCF2 was performed in the presence of the AlexaFluor 647-conjugated anti-Flag mAb M2 at a 1:100 final dilution. Enzymatic cleavage of CCF2 by β-lactamase in the target cells was analyzed by flow cytometry (FACSCanto, BD Biosciences).