Deae dextran hydrochloride

Dilutions (10-fold) of supernatants were prepared in Opti-MEM and 150 μL per well was used to infect confluent MDCK cells in 12-well dishes, in duplicates, for 1 h at 37°C. Virus inoculum was then removed, and cells were overlaid with 1 mL MEM (Gibco, 61100053) containing 0.6% oxoid agar (Thermo Scientific, LP0028), 1 μg/mL TPCK trypsin, 0.01% DEAE-Dextran hydrochloride (Sigma Aldrich, D9885), and 0.5% NaHCO3 (MP Biomedicals, 194553). Cells were collected at 48 hpi, fixed with 4% paraformaldehyde (PFA), and plaques were visualized by crystal violet staining.

MuLV replication was assessed by transfection of proviral clone constructs and subsequent infection of Rat2 cells with virions released from transfected cells. 293T monolayers at 40% confluence were prepared in six-well plates and transfected with pNCA or a mutant derivative. To assess virus production efficiency, cells were harvested at 72 hpt and subjected to SDS-PAGE and immunoblotting for viral proteins. A cellular protein (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) was used as a loading control. Tissue culture supernatants were filtered through a 0.45-μm-pore-size filter prior to infection of fresh monolayers or biochemical analysis. Filtered supernatant (500 μl) from transfected cells was added directly to six-well plates of Rat2 monolayers at 10% confluence and incubated for 1 h at 37°C, and the volume was adjusted to 3 ml with fresh medium (DMEM with 2% FCS) in the presence of 30 μg/μl of DEAE-dextran hydrochloride (Sigma). Cells were incubated at 37°C and 10% CO₂ for 4 days. Cells were harvested directly in Laemmli's sample buffer, and proteins were analyzed by immunoblotting. Viral supernatant was sterile filtered and used for protein and RNA analysis.

MuLV replication was assessed by transfection of the proviral clone and subsequent infection of Rat2 cells with virions released from transfected cells. 293T monolayers at 40% confluence were prepared in six-well plates and transfected with pNCA. At 72 h p.t. tissue culture supernatants were filtered through a 0.45-µm-pore-size filter prior to infection of fresh monolayers. Filtered supernatant (3 ml) from transfected cells was added directly to 10-cm plates of Rat2 monolayers at 10% confluence and incubated for 1 h at 37 °C, and the volume was adjusted to 10 ml with fresh medium (DMEM with 2% FCS) in the presence of 30 µg/µl of DEAE-dextran hydrochloride (Sigma). Cells were incubated at 37 °C and 10% CO₂ for 4 days.

Cloned viral Envs were used to generate pseudoviruses by co-transfection with pSG3 plasmid of HEK-293T cells as indicated above and tested in TZM-bl cells to determine the infectivity capacity. Serial dilutions of the pseudoviruses generated with the different Envs of the different groups of patients were made in a 96-well plate. Then, 1 × 10⁵ TZM-bl cells were added to the pseudoviruses with DEAE dextran hydrochloride (Sigma) at 18 μg/ml. After 48 h of incubation at 37°C, luciferase activity was measured (Fluoroskan Accent, Labsystems) using Brite-Lite (PerkinElmer). Uninfected TZM-bl cells were used as a negative control. The TCID₅₀ (Median Tissue Culture Infectious Dose) value was calculated with Montefiori template and normalized with the viral concentrations (summarized in the scheme of Figure 1D).

Virus quantification in the culture supernatants was done by enzyme-linked immunosorbent assay (ELISA) for SIV p27 antigen using a commercial kit (Advanced Bioscience Laboratories, ABL, Rockville, MD, USA) following the manufacturer’s instructions. Supernatants with >10 ng/mL SIV p27 were pooled to make a single virus stock. The infectivity of the virus stock was assessed and the TCID₅₀ was determined in TZM-bl cells. To do this, the virus stock was inoculated at 1:5 serial dilutions in quadruplicate wells of 96-well plates containing in TZM-bl cells in the presence of 20 µg/mL of DEAE-dextran hydrochloride (Sigma-Aldrich, St. Louis, MO, USA). Virus titre was determined 48 h post-infection by measuring the level of luciferase activity expressed in infected cells. The TCID₅₀ was calculated as the dilution point at which 50% of the cultures were infected.

DMEM, RPMI, fetal bovine serum (FBS) and l-glutamine were purchased from Gibco Life Technologies Ltd., Burlington, Ontario, Canada. Penicillin/streptomycin, phosphate buffered saline (PBS) were from Wisent, St. Bruno, Quebec, Canada. Human recombinant IL-1β was product of R&D Systems (Minneapolis, MN, USA), and LPS endotoxin (Escherichia coli, serotype O128:B12) and DEAE-Dextran hydrochloride of Sigma (Oakville, Ontario, Canada), while murine IL-4 was from Peprotech, Quebec, Canada. Antibodies used in this study were: COX-2 rabbit polyclonal antibody from Thermo Fisher Scientific (Burlington, Ontario, Canada), and COX-2 (C-20) goat polyclonal, NFκB p65 (C-20), p50 (H-119) and Lamin A (H-102) rabbit polyclonal antibodies from Santa Cruz (Dallas, TX, USA). ESE-1 rabbit monoclonal antibody was produced in our laboratory in collaboration with Epitomics, Burlingame, CA, USA [22 (link)]. Hsp90 rabbit polyclonal and β-actin mouse monoclonal antibodies were purchased from Cell Signaling Technology (Whitby, Ontario, Canada).