2.2 VSV pseudotyped virus preparation and transduction
HEK293T cells were transfected with either the pcDNA3.1-RABV-GP,
pHEF-VSV-G, or pcDNA3.1 empty vector. For transfection, plasmid DNAs
were incubated with polyethyleneimine (PEI; Polysciences, Inc.,
Warrington, PA, USA) at 1:3 DNA:PEI ratios in Opti-MEM (Life
Technologies, Carlsbad, CA, USA) for 15 min at room temperature, and
then added dropwise to the adherent cells (2 µg DNA per
106 cells). At 24 h post-transfection, cells
transduced with VSVluc-VSV∆G were complemented with Junin virus
glycoproteins for 1 h. Cell-free supernatants were collected at 24-72 h
post-transduction and filtered through a 0.45 µm filter. The pseudotyped
viruses in the supernatants were pelleted by centrifugation at 10,000 ×
g for 10-18 h, suspended in phosphate-buffered saline (PBS, pH 7.4), and
kept at −80 °C until use.
For transduction, BHK21 cells were incubated with pseudotyped viruses at
normalized inputs, based on VSV M protein levels, for 1 h at 37 °C.
Where indicated, mouse anti-C9 (Santa Cruz) or monoclonal mouse
anti-rabies virus (Rab-50, Santa Cruz) were added during virus
absorption. At 18 h post-transduction, cells were lysed, and luciferase
levels were quantified as a measure of VSV pseudotyped virus entry.