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Cells: Human bronchial epithelial cells (BEAS-2B; ATCC CRL-9609), human embryonic kidney cells (HEK-293T; ATCC CRL-3216), baby hamster kidney cells stably expressing T7 polymerase (BSR-T7/5) and African green monkey kidney epithelial cells (Vero; ATCC CCL1-81) were maintained at 37o_{C in 5% CO}

2 in Dulbecco’s modified Eagle’s medium

(DMEM) supplemented with 7.5% fetal bovine serum. The BSR-T7/5 cell line was supplemented with G-148 every 5th _{passage and cells were transfected using GeneJuice}

(Novagen) according to the manufacturer’s instructions.

Molecular Biology: Point mutations of escape mutants from adaptations were introduced via the QuickChange mutagenesis PCR protocol. Recombinant viruses were produced by

introduction of point mutations in a shuttle vector that used restriction sites mluI and NheI to transpose the G ORF to a vector containing full length genomic DNA.

Compounds:The screening set contained a compilation of commercially available libraries (ChemDiv, 30,000 compounds; ChemBridge, 100,000 compounds; and proprietary collections from the Emory institute for drug development, 1,155 compounds; and Kansas University,11,520 compounds) that are specifically curated against compounds with pan-assay interfering or other undesirable chemical structures. The compounds from each library were

dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 µM and stored at -80o_{C. All}

HTS electronic compound management, data storage, and data analysis was performed using the MScreen software package. Prior to screening, all compounds were inventoried in Mscreen and reformatted into barcoded 384-well plates using a Nimbus96 liquid handler (Hamilton Robotics). Each 384-well daughter plate consists of positive (cycloheximide) and negative (DMSO)

controls in a checkerboard pattern on the two lateral columns on both sides of every plate.

HTS Protocol: BEAS-2B cells (3.5x103_{/well) were seeded in barcoded 384-well white}

wall/clear bottom plates using a MultiFlo automated dispenser (BioTeK) equipped with dual 10 µl peristaltic pump manifolds followed by collection (150 x g for 90 sec at 25o_{C) and incubated}

for 14 hours at 37o_{C in 5% CO}

2. Compound addition for a final concentration of 5 µM (20

nl/well) was performed by the Nimbus96 liquid handler outfitted with a high-density pintool (V&P Scientific) attached to the pipetting head. Both source and assay plates were read by the Nimbus96 unit at the time of compound stamping. Co-infection of recRABVDG-SBNG

(multiplicity of infection (MOI) = 0.01) at 10 µl/well was performed using a Multiflo dispenser followed by spin collection and incubation at standard conditions for 48 hours post-infection. Plates were loaded into an H1 synergy plate reader (BioTek) with a stacker and identified with an integrated barcode reader, that also automatically injected nano-Glo substrate (10 µl/well) and bioluminescence was recorded 3 minutes after substrate addition.

HTS Data normalization and Hit identification: Data analysis was performed using raw data imported from the Nimbus96 (source and assay barcode maps) and plate reader bioluminescence readouts to the Mscreen software package. Normalized relative inhibition values were calculated by dividing the results by the difference between positive and negative plate control means. Robust z-scores were calculated by the formula: robust z-score = (Si –

median (Sall))/MAD(Sall) and MAD(Sall) = 1.4826 × median (|Si – median (Sall)|) (where Si = individual compound value, and Sall= values of all compounds on the plate). Hit candidates were defined as compounds with ³ 70% inhibition of normalized signal intensity against

recRABVDG-SBNG, a robust z-score ³2.0, and <35% inhibition against RSV-FF, WSN-nano, and HPIV-3-nano. Hit candidates were also queried using the SciFinder database package to determine known bioactivity.

Automated dose-response counter-screening: BEAS-2B cells (3.5 x 103 _cells/well)

were seeded in barcoded white wall/clear bottom 384-well plates using a MultiFlo automated dispenser as described above. The Nimbus96 unit was used to prepare 3-fold dilutions hit compounds (0.078 – 10 µM) in barcoded 384-well plate format for subsequent dose-response and cytotoxicity counter-screening. BEAS-2B cells (3.5 x 103_{/well) were seeded in barcoded}

white wall/clear bottom 384-well plates using a MultiFlo automated dispenser as described above, then stamped with the counterscreen plates using the high-density pin tool. Cell viability in the presence of compound was tested using PrestoBlue substrate (5 µl/well)

(LifeTechnologies) 48 hours post compound addition. Addition of PrestoBlue was performed according to the manufacturer’s instructions and read using the H1 synergy plate reader (excitation at 560 nm, emission at 590 nm and gain of 85).

Dose response reporter Assays: The Nimbus96 unit was used to transfer three-fold or five-fold serial dilutions (as indicated by the Figure) of hit candidate compounds to 96-well plates seeded with BEAS-2B, 293T, or BSR-T7/5 cell lines (1.5 x 104 _{cells/well). Controls}

included four positive (1 mg/ml cycloheximide) and four negative (0.05% DMSO) wells on each plate. The cells were infected with recRABVDG-SBNG (MOI = 0.1) using the MultiFlo

hours post infection. Dose-response inhibition curves were generated according to the formula: % inhibition = (XSample – XMin)/(XMax – XMin) × 100, with XMin representing the average of the

positive and XMaxthe average of the negative control wells and the Prism (GraphPad) nonlinear

regression function with a four-parameter regression slope was applied to determine 50% effective concentration (EC50) of each hit compound. All dose-response activity assays were

performed in 3 independent repeats.

Kinetic cell-cell fusion assay: BEAS-2B cells (1.5 x 105 _{cells/well) were cotransfected}

with 1.0 µg RABV-G or RABV-Gescapemutant expression plasmids and 0.5 µg DSP1-7 expression

plasmid. DSP8-11 was transfected independently. Cells were harvested 24 hours post- transfection and reseeded with equal parts of G and DSP1-7 containing cells with DSP8-11

containing cells in a 96-well plate to a total of 4 x 103 _{cells/well. Cells were pretreated with}

viviren substrate 4 hours post-reseeding for 1.5 hours according to the manufacturer’s

instructions. Fusion was triggered by the addition of 100 µl of DMEM with pH=6.0 and ViviRen signal was measured by the Cytation5 cell imaging multimode reader (BioTek) every 30 minutes for 4 hours. Values are shown as percent normalized to RABV-G and are based on 3 independent repeats.

Virus Adaptation: BEAS-2B cells were infected with recVSV-GFP-RABV-G with an MOI of 0.1 TCID50 units/cell and incubated for 30 min at 37oC 5% CO2 before addition of 1.1 µM of GRP-60367. Fresh BEAS-2B cells were infected with 10-fold dilution of supernatant- associated virions every two days with gradual increase of compound until 100 µM was tolerated. Total RNA was extracted from 8 individually adapted clones and cDNAs were

generated using random hexamer primers, and the G protein ORF was amplified and subjected to Sanger sequencing. Confirmed escape mutations were introduced to G protein expression

plasmids and also rebuilt into the recVSV-GFP-P plasmid background for recovery of escape mutants in the context of full-length virus.

Recombinant Virus Recovery: RecVSV-RABV-G were recovered as previously described [211]. Briefly, plasmid vectors expressing VSV-N (0.5 µg), VSV-P(0.4 µg), VSV-L (0.2 µg) and plasmid with genomic recVSV-RABV-GFP or recVSV-RABV-G-GFP mutants (1.0 µg) were transfected in BSR-T7/5 cells (2.5 x 105 _{cells/well) and incubated at 37}o_{C and}

monitored for viral particle formation by visualized GFP signal. Viral supernatant was overlaid onto fresh Vero-E6 cells infection was allowed to proceed and viral supernatant was collected and TCID50 was quantified by GFP signal.

Surface biotinylation, SDS-PAGE and immunoblotting: BSR-T7/5 cells were seeded in a 6-well plate (4 x 105 _{cells/well) and transfected with 1 µg of RabV G or RABV G adaptation}

mutants. Twenty-four hours post-transfection cells were washed twice in cold PBS, cells were then biotinylated with 0.5 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (Pierce) for 30 minutes, quenched with 1M Tris (pH 7.5), and washed three times with cold PBS prior to lysis in RIPA buffer (1% sodium deoxycholate, 1% nonidet P-40, 150 mM NaCl, 50 mM Tris-Cl at pH 7.2, 10 mM EDTA, 50 mM NaF, and protease inhibitors (Pierce)). Strepdavidin bead slurry was added to each cleared lysate sample (14,000 rpm for 30 minutes at 4o_{C) and}

incubated on a rotor for 2 hours in 4o_{C. Washed precipitates and total lysates were fractioned by}

SDS-PAGE and transferred onto a PVDF membrane by semi-dry transfer. Proteins were detected by immunoblot with anti-RABV G antibodies derived from rabbit sera followed by