Chloroquin

HEK293T cells (ATCC, No.CRL‐112689) were cultured for at least 1 week in medium consisting of DMEM high glucose (Sigma‐Aldrich, USA), 10% heat‐inactivated fetal‐calf serum (FCS, PAN‐Biotech, Germany), 4 mM l‐glutamine (Lonza), 200 U/mL penicillin, and 200 μg/mL streptomycin (Thermo Fisher Scientific), and 1 mM sodium pyruvate (Lonza). Addition of 1% nonessential amino acid (Thermo Fisher Scientific) and 1% Nutridoma (Roche, Switzerland) are optional. HEK293T cells were seeded in cell culture plates (Greiner Bio‐One, Austria) and treated with 20 mM Chloroquin (Sigma‐Aldrich) the next day prior to calcium‐phosphate transfection with plasmids encoding light and heavy chains of anti‐Langerin‐EBNA1 fusion protein. The day after transfection, the supernatant was carefully removed, the adherent HEK293T cells were washed with PBS (Thermo Fisher Scientific), and incubated for 2 days in a fresh medium consisting of DMEM high glucose, 4 mM l‐glutamine, 100 U/mL penicillin/100 μg/mL streptomycin, 1 mM sodium pyruvate, 1% nonessential amino acid, and 1% Nutridoma. The supernatant was collected, fresh medium was added to the cells for additional 3 days of culture. Supernatants from the two time points were pooled, centrifuged, sterile filtered (Stericup 0.22 μm; Merck, Germany), checked for a pH of 7, and stored at 4°C until further use.

For the production of eGFP labeled pseudovirus, HIV-1-derived plasmids pCHIV and pCHIVeGFP [52 (link)], which express all proteins from HIV-1NL4-3 except for Nef, were used. Transfection of these plasmids yields non-infectious eGFP labeled particles. 5–6 × 10⁶ HEK293T cells were seeded into 10 cm tissue culture dishes one day prior to transfection. Shortly before transfection, medium was exchanged with DMEM without FCS supplemented with chloroquin (final concentration 25 µM, Sigma-Aldrich, Darmstadt, Germany). The transfection mixture contained 10 μg pCHIV and 10 μg pCHIVeGFP, 50 μL 2.5 M CaCl₂ (Carl Roth GmbH, Karlsruhe, Germany) and 450 μL H₂O. Derivatives of these plasmids carrying a 2 bp insertion at the NdeI site, resulting in a frameshift in the viral env gene were used to prepare ΔEnv control particles. For transfection, the mixture was added to 500 μL 0.2 M HEPES buffer, pH 7.05 (Sigma-Aldrich, Darmstadt, Germany) and incubated for 20 min at room temperature. The transfection mixture was added to the prepared 293T cells and cultured for 6 h before medium exchange. The supernatant was collected after 12 and 36 h, then filtrated and concentrated by overnight centrifugation at 6000 rpm and 4 °C. The supernatant was discarded, and the pellet was resolved in 100 µL of PBS. The particle concentration was quantified by p24 ELISA (Innogenetics, Gent, Belgium).