Fig 1.
FASN inhibition reduces yield of RSV A, PIV3 and HRV infectious progeny.
Experiments were performed as shown in the diagram in (A). (B) A549 cells were infected with RSV A2 GFP (MOI 0.1). (C) HEp2 cells were infected with PIV3 (MOI 0.001). (D) HeLa cells were infected with HRV16 (MOI 0.0002). Progeny virus harvested from cells infected for 72 hours in the presence or absence of the FASN inhibitors TVB-3166 or TVB-2722 was used to infect Vero cells. Progeny was titered by automated microscopy (B) or plaque assay (C, D). (E) Infected or mock-infected cells were treated as indicated and cell viability was assessed 72 hours post treatment by Cell Titer Glo assay. Graphs represent the mean ± standard deviation of results from duplicate samples (* = p<0.05, ** = p<0.01, *** = p<0.001).
Fig 2.
FASN inhibitors reduce infectious progeny of multiple RSV strains.
A549 cells were infected at MOI 0.1 with RSV strains A Long (A), ATCC 2012–10 (B), B-WV/14617/85 (C), or B 18537 (D). Progeny virus harvested from cells infected for 72 hours in the presence or absence of the FASN inhibitor TVB-3166 was used to infect Vero cells. Progeny was titered by plaque assay. Graphs represent the mean ± standard deviation of results from duplicate samples (* = p<0.05, ** = p<0.01).
Fig 3.
Anti-RSV activity of TVB-3166 is mediated by palmitate reduction.
The fatty acid synthesis and utilization pathway is shown in (A). (B) A549 cells were infected with RSV A2 GFP (MOI 0.03) followed by treatment with TVB-3166 or the RSV fusion inhibitor JNJ-2408068 in the presence or absence of 50 μM palmitate-BSA (PA). Infection levels were quantified 72 hours post infection by automated microscopy. Graphs represent the mean ± standard deviation of results from duplicate samples (* = p<0.05, ** = p<0.01).
Fig 4.
Inhibition of FASN by TVB-3166 reduces RSV spread and intracellular levels of RSV protein and RNA.
A549 cells were infected with RSV A2 GFP at MOI 0.1 (A, B, D) or MOI 2 (C), then treated with DMSO or TVB-3166, and harvested at the indicated times post infection. (A) Cells were fixed and imaged at 10X magnification by fluorescent microscopy. (B) Percent infection was calculated using automated imaging. (C) RSV F and G protein levels were detected by Western blot. (D) RSV F RNA levels were determined by qPCR after reverse transcription with oligo-dT or genomic primers and normalized to -actin. qPCR was performed in duplicate from duplicate samples and all C(t) values were normalized to the lowest of the four DMSO-72h C(t) values. Graphs represent the mean ± standard deviation of results from four samples (** = p<0.01, *** = p<0.001, **** = p<0.0001).
Fig 5.
TVB-3166 reduces infectivity of progeny virions.
A549 cells were infected with RSV A2 GFP at MOI 0.1 and treated with DMSO or TVB-3166 as indicated. (A) Cells were fixed at 72 hpi and percent infection was calculated using automated imaging. (B-D) Infected cell media were collected at 72 hpi and treated with RNase A. (B) RNase-protected RNA was extracted from cell media and reverse-transcribed with genomic primers, and RSV N RNA levels were determined by qPCR. (C) Progeny virus harvested from cell media was used to infect Vero cells and progeny was titered by automated microscopy. (D) Infectivity of progeny virions is expressed as PFU per million RNA copies. Graphs represent the mean ± standard deviation of results from duplicate samples (* = p<0.05, ** = p<0.01, *** = p<0.001).
Fig 6.
Progeny virions from TVB-3166-treated cells fail to replicate in naïve cells.
A549 cells were infected with RSV A2 GFP at MOI 0.3 and treated with DMSO or 0.2 μM TVB-3166 as shown in (A). Infected cell media were collected at 72 hpi and treated with RNase A. RNase-protected RNA was extracted from cell media and reverse-transcribed with genomic primers, and RSV progeny particle numbers (RSV N RNA levels) were determined by qPCR. Equal particle numbers of DMSO or TVB-3166 progeny virus were allowed to attach to Vero cells at 4°C, following which cells were shifted to 37°C to allow viral entry, and treated with trypsin to destroy un-internalized virus. Internalized virus was quantitated by qPCR. Viral attachment is shown in (B); entry and replication in (C). Graphs represent the mean ± standard deviation of results from duplicate samples (* = p<0.05, **** = p<0.0001).
Fig 7.
TVB-3166 exhibits both prophylactic and therapeutic anti-RSV effects in BALB/c mice.
(A) Twice daily treatment with TVB-3166 (p.o.), Ribavirin (i.p.) or drug vehicle (p.o.) was begun 2 hours after intranasal inoculation of mice with 5.5 x 105 PFU of RSV Long. One group received vehicle on day 0 and TVB-3166 on subsequent days. Lungs and broncheoalveolar lavage (BAL) fluid were harvested five days post infection. (B) Lung titers were determined by plaque assay on HEp2 cells. (C) Percent change in initial body weight was measured daily. (D) Leukocyte populations in BAL were quantified by differential count. Graphs represent the mean ± standard deviation of results from groups of 5 or 10 animals (* = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001).