Supplemental Methods
One-step growth curve - Cells were infected with the indicated viruses at an MOI of 3 pfu/cell
for 1 hour. Cells were then washed with PBS and incubated at 37°C. 100 μl aliquots were taken
at time 0, 4, 8, 12, 24, and 48 hours post infection and titers assessed with a standard plaque
assay.
Titration of single cycle MG1-Gless – 2x104 vero cells were seeded in 96 well plates and
incubated at 37°C in 5% CO2 for 24 hours. Cells were infected with 50ul of 10-fold serial
dilutions of MG1-Gless for 2 hours, then replaced with 100ul complete DMEM. 24 hours later,
GFP+ cells were counted with a fluorescent microscope.
MG1-Gless Virus
MG1-Gless was cloned based on wild type G-deficient Maraba virus. Briefly, PCR was carried
out with 100 ng of mutagenic primer and 100 ng DNA template (wild type G-deficient Maraba
virus) with Hot Start addition of enzyme and typical PCR setup (98°C-10 seconds, 60°C-30
seconds, 72 °C for 7.5 minutes for 33cycles). The parental plasmid was digested with DpnI
(NEB) (37 °C for 1 hour) and 10 μl of the 25 μl DpnI-digested PCR mixture was used to
transform TOP-10 competent cells (Invitrogen, Carlsbad, CA). Positive clones were screened by
introduction of noncoding change restriction site changes followed by sequencing. The mutant
described here is Leu-123 to Trp in the M protein (L123W).
MG1-Gless virus rescue
24 hours post A549 cell seeding (3.0 × 105 cells/well in 6-well plates), vaccinia virus (MOI 10)
expressing the T7 RNA polymerase in OptiMEM medium was used for 1.5 hours of infection.
Following removal of vaccinia virus, each well was transfected with LC-KAN Maraba (2ug)
together with pCI-Neo constructs encoding for Maraba N (1ug), P (1.25ug), L (0.25ug) and G
(0.25ug) with lipofectamine 2000 (5ul/well) according to the manufacturer’s instructions. The
transfection reagent was removed 5 hours later and replaced with cDMEM. At 48 hours post
transfection, medium was filtered (0.2um) to remove contaminating vaccinia virus, and 1 ml was
used to infect 293FT (pre-transfected with G) cells in each well of a 6-well plate. Cytopathic
effects and GFP visible 24-48 hours later were indicative of a successful rescue, which was
confirmed by purifying viral RNA and reverse transcriptase (RT)-PCR with Maraba-specific
primers. T-REx™-293 (G gene was cloned into this cell line to conditionally supply G protein)
was used to manufacture MG1-Gless virus. Lastly, purification on OptiPrep™ Density Gradient
Medium was performed.
Immunohistochemistry (IHC) analysis of lung tumor tissue
IHC was performed on formalin-fixed, paraffin-embedded 5μm mouse lung tissue sections. The
anti-VSV antibody recognizing viral proteins was used at a dilution of 1:250 for 1 hour.
Microwave heating in a solution of sodium citrate (pH 6) was performed prior to incubation with
the primary antibody. Negative control slides were prepared with lung tissue from uninfected
B16lacZ tumor bearing mice.
Positive control slides were prepared from pancreatic tumor
bearing mice receiving intratumoral injections of virus.