VSV-G-driven Host Cell Entry

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)].