Plate luminometer

To analyze VSV-G-driven host cell entry, VSV vectors were pseudotyped with either VSV-G wt or mutants A133R and LXXXL. For this, 293T cells were transfected with the respective expression plasmids or empty plasmid as negative control. At 24 h post transfection, cells were inoculated with VSV-G trans-complemented VSV*ΔG that lacks the genetic information for VSV-G but codes for eGFP and firefly luciferase from two independent transcription units [42 (link)] (kindly provided by G. Zimmer) at an MOI of 3. At 1 h post infection, the inoculum was removed and the cells were washed with PBS. Next, medium containing a neutralizing antibody directed against VSV-G (I1, produced from CRL-2700 hybridoma cells, ATCC) was added to the cells and left for 1 h in order to neutralize residual input virus that had not entered the cells so far. Subsequently, the cells were again washed and further incubated with fresh culture medium for 16–18 h. Then, the supernatant was collected, clarified from cellular debris by centrifugation (4,700 rpm, 10 min, 4°C) and used for transduction experiments. For this, 293T cells were inoculated with identical volumes of the respective VSV pseudotypes and firefly luciferase activity in cell lysates was quantified at 20 h post inoculation using a plate luminometer (Hidex) and commercial substrates (PJK and Promega) as described elsewhere [43 (link)].

293T cells were seeded in 12-well plates at 1.5 × 10⁵ cells per well and co-transfected via calcium phosphate precipitation with 1 µg of a plasmid encoding the HIV-1 NL4-3 proviral genome and plasmids expressing SFL, SFLS, or SFL mutants. The following day, the medium was replaced and 1 mL of fresh DMEM was added to the cells. After 48 h, virus-containing supernatants were collected and centrifuged for 5 min at 2800× g to remove cell debris. Next, TZM-bl cells seeded in a 96-well plate at 1.5 × 10⁴ cells per well were infected with equal volumes of supernatants (diluted at 1:3 in the medium) in technical triplicates. At 48 h post-infection, TZM-bl cells were lysed with PBS/0.5% Triton-X 100 containing lysis buffer for 30 min at room temperature. Next, 45 µL of cell lysate was transferred into a white opaque-walled 96-well plate and after adding 45 µL of FLuc substrate (PJK, Kleinbittersdorf, Germany), infection efficiency was determined by measuring luminescence signals using a plate luminometer (Hidex, Turku, Finland).

We employed a previously described protocol for the generation of VSV pseudotype particles (VSVpp) that is based on a replication-deficient VSV vector that lacks the genetic information for VSV-G but instead contains the genetic information for eGFP and firefly luciferase (fLuc) as reporters of transduction efficiency (VSV*ΔG-fLuc, kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern/Switzerland) [37 (link),40 (link)]. In brief, 293T cells transfected with expression vectors for MERS-CoV S, VSV-G (positive control) or empty expression vector (negative control) were inoculated with VSV*ΔG-fLuc for 1 h before being washed with PBS and further incubated for 16 h with culture medium that was supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) (except for cells expressing VSV-G). The produced VSVpp were inoculated onto BHK-21 cells expressing WT or mutant DPP4, or no DPP4 (empty expression vector, negative control) and incubated for 16–18 h before fLuc activity in cell lysates was quantified as an indicator for transduction efficiency using the Beetle-Juice kit (PJK) and a plate luminometer (Hidex) [41 (link)].