1. Volume 20, Issue 2, Pages 167-177 (August 2016)
Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses 
Bryan C. Mounce, Enzo Z. Poirier, Gabriella Passoni, Etienne Simon-Loriere, Teresa Cesaro, Matthieu Prot, Kenneth A. Stapleford, Gonzalo Moratorio, Anavaj Sakuntabhai, Jean-Pierre Levraud, Marco Vignuzzi 
Cell Host & Microbe 
Volume 20, Issue 2, Pages (August 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 20, 167-177DOI: (10.1016/j.chom.2016.06.011)
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3. Figure 1 SAT1 Is Upregulated by Type I IFN Stimulation
(A) The polyamine pathway and relevant enzymes and inhibitors used in this study.
(B–F) Huh7 cells were stimulated with 10,000 U/mL IFNβ for 24 hr and harvested for mRNA expression of the IFN-stimulated genes (B) ISG15 and (C) viperin, as well as polyamine biosynthetic enzymes (D) SAT1, (E) ODC1, and (F) OAZ1.
(G) Western analysis of expression of SAT1 and ODC1 in Huh7 cells stimulated with 10,000 U/mL IFNβ or 10 μM DENSpm for 16 hr.
(H) Western and chromatographic analysis of SAT1 knockout cells and controls after 24 hr IFNβ treatment, probing for SAT1 (above) and polyamines (below).
(I) SAT1 knockout cells and unmodified controls were treated with IFNβ for 4 hr prior to infection with CHIKV. Viral titers were determined at 48 hpi.
∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ using Student’s t test (n ≥ 3). Error bars represent ± 1 SEM. Western blots are representative of at least three independent experiments.
Cell Host & Microbe  , DOI: ( /j.chom )
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4. Figure 2 SAT1 Is Antiviral
(A) SAT1 and mutant constructs were co-transfected into BHK-21 cells with CHIKV RNA, and viral titers were measured after 24 hr (above). Western analysis of transfected cells prepared in parallel is shown below.
(B–D) (B) BHK-21 cells were transfected with SAT1 constructs and infected 24 hr later with CHIKV and ZIKV at an MOI of 1. Cells were fixed and stained at 16 hpi for ZIKV and 6 hpi for CHIKV with antibodies against SAT1 (green) and virus (red). Stained cells were analyzed for overexpression of SAT1 and viral replication and the percent infected was quantified for CHIKV and ZIKV (right). SAT1-inducible cell lines were generated and treated with 4 μg/mL tetracycline 16 hr prior to infection with (C) ZIKV and (D) CHIKV. Titers were measured at 24 hpi.
(E) Western analysis of cells induced in parallel to (C) and (D).
(F) Vero-E6 cells were treated for 16 hr with escalating doses of DENSpm and infected with ZIKV at MOI 0.1, and viral titers were measured at 24 hpi.
(G) Transcript levels of SAT1 were measured by qPCR and calculated as fold induction over untreated controls (above), and protein levels of SAT1 were measured by western blot of stimulated cells at 16 hr post-treatment (below).
∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ using a one-tailed Student’s t test (n = 3). Error bars represent ± 1 SEM. Western blot is representative of two independent analyses. See also Figure S1.
Cell Host & Microbe  , DOI: ( /j.chom )
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5. Figure 3 Polyamines Facilitate Alpha- and Flavivirus Replication
(A) BHK-21 cells were pretreated for 4 days with 500 μM DFMO to deplete polyamines and infected with CHIKV at MOI 0.1, and viral titers measured by plaque assay on Vero-E6 cells. Exogenous polyamines were added at the time of infection (gray circles).
(B) Polyamines were examined by thin-layer chromatography of cellular extracts treated in parallel to (A).
(C) DFMO-treated BHK-21 cells were infected with ZIKV for 24 hr and titers measured by focus-forming assay on C6/36 cells.
(D) BHK-21 cells were treated with 500 μM DFMO for the length of time indicated, analyzed for polyamine content (above), and infected with CHIKV at MOI 0.1 for 24 hr.
(E and F) BHK-21 cells were pretreated for 4 days with escalating doses of DFMO, infected at MOI 0.1 with (E) CHIKV and (F) ZIKV, and titers analyzed 24 hpi.
(G) BHK-21 cells were pretreated with 500 μM DFMO and increasing concentrations of exogenous polyamines were added at time of infection. Titers were determined 24 hpi. Lower dashed line indicates the average titer from DFMO-treated cells; upper dashed line indicates the average titer from untreated cells.
(H) BHK-21 cells were pretreated with 500 μM DFMO and polyamines were added at time of infection. Titers were determined 24 hpi. Comparisons were made between viral titers from treated and untreated cells.
∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ using a one-tailed Student’s t test (n ≥ 3). Error bars represent ± 1 SEM. See also Figures S2–S4.
Cell Host & Microbe  , DOI: ( /j.chom )
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6. Figure 4 Polyamines Stimulate Viral RNA Synthesis
(A and B) BHK-21 cells were pretreated with 500 μM DFMO and infected at MOI 0.1. RNA was quantified by qRT-PCR with primers against (A) CHIKV and (B) ZIKV.
(C) Membranes derived from CHIKV-infected cells were used for membrane-based replication assays to monitor generation of nascent genomes over 45 min (above). Viral RNA was incubated with membranes derived from uninfected cells as a control for RNA stability (below).
(D) Quantitation of genomes as shown in (C).
(E) Membranes were incubated with individual polyamines as in (C) and nascent viral genomes were quantified after 45 min.
(F) BHK-21 cells were treated with polyamines and immediately infected with CHIKV at MOI 1. Viral RNA was measured as indicated.
∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ using a one-tailed Student’s t test (n = 3). Error bars represent ± 1 SEM.
Cell Host & Microbe  , DOI: ( /j.chom )
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7. Figure 5 Viral Translation Relies on Polyamines
BHK-21 cells were pretreated with 500 μM DFMO and transfected with 1 μg CHIKV genomic RNA.
(A and B) (A) Viral titers were measured after 24 hr by plaque assay and (B) viral genomes were measured at 2 hr by qRT-PCR.
(C–E) (C) Pretreated cells were transfected with RNA-encoding luciferase to determine translational levels of transfected transgenes, and luciferase activity was measured after 24 hr. Pretreated cells were transfected with replication-deficient (D) CHIKV and (E) dengue virus containing luciferase within the viral genome, and luciferase activity was measured after 24 hr. Dashed line indicates background luciferase activity in non-transfected cells.
∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ using a one-tailed Student’s t test (n ≥ 3). Error bars represent ± 1 SEM.
Cell Host & Microbe  , DOI: ( /j.chom )
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8. Figure 6 Polyamines Facilitate Alphavirus Infection In Vivo
(A) Drosophila melanogaster were fed DFMO in sucrose solution for 7 days before SINV infection.
(B) D. melanogaster expressing siRNA against ODC2 were infected with SINV and titers were determined.
(C) Danio rerio were treated with DFMO for 4 days before SINV infection. Titers were determined at 24 and 48 hpi.
(D–F) Polyamine analysis on (D) DFMO-treated D. melanogaster, (E) wild-type and mutant D. melanogaster, and (F) DFMO-treated D. rerio.
(G) D. rerio was fixed and visualized for nuclei (DAPI, blue), neurons (AcTubulin, red), and SINV (green) at 24 and 48 hpi.
(H) Working model for the role of polyamines in the type I IFN response and viral replication.
∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ using a one-tailed Student’s t test (n ≥ 3). Error bars represent ± 1 SEM.
Cell Host & Microbe  , DOI: ( /j.chom )
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