SINE compounds inhibited New World alphaviruses more dramatically than Old World alphaviruses.

<p>(A) Capsid alignment using Uniprot. Asterisks indicate positions which have a fully conserved residue. Colons indicate conservation between amino acids of strongly similar properties. Periods indicate conservation between amino acids of weakly similar properties. Residues within the consensus NES sequence (ФxxxФxxФxФ, where Ф = L, I, F, V or M and x = any amino acid) are highlighted in yellow. Positively charged residues within the NLS are highlighted in green. The supraNES sequence identified in VEEV capsid is indicated [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005122#pntd.0005122.ref017" target="_blank">17</a>]. Conservation among different strains of the four viruses aligned—VEEV-TrD (GenBank Accession Number AAB0251), VEEV-TC83 (CAA27883), EEEV-82V-2137 (CAA29261), and WEEV-BFS1703 (AAA42999)–was strong for the sequence examined. (B) Vero cells were treated for two hours prior to infection with DMSO (0.5%), 45 nM Leptomycin B, or 2.5 μM of KPT-185, KPT-301, KPT-335, or KPT-350. After an hour infection with EEEV (GA97) (MOI 1), the cells were post-treated with inhibitors. At 16 hpi, supernatants were collected and plaque assays performed using Vero cells. Graphs represent biological triplicates and each experiment was performed twice. (C) Vero cells were treated as described above, infected with WEEV (California 1930) (MOI 1), and titered by plaque assay in Vero cells. (D) Vero cells were treated as described above, infected with SINV (EgAr 339) (MOI 1), and plaque assays were performed using BHK-21 cells. (E) Vero cells were treated as described above, infected with CHIKV (S27) (MOI 1), and plaque assays were performed using BHK-21 cells. *p-value ≤ 0.001, **p-value ≤ 0.005 (compared to DMSO treated cells at the corresponding time point).</p

SINE compounds reduced VEEV viral replication in a dose-dependent manner.

<p>(A) For two hours prior to infection Vero cells were treated with 2.5 μM of KPT-185, KPT-301, KPT-335, KPT-350, DMSO (1%), or 45 nM Leptomycin B. Cells were infected with VEEV-TC83luc (MOI 1), and cells post-treated after infection. At 16 hpi, the BrightGlo Luciferase Assay was performed. Data are the averaged luminescence of biological triplicates represented as a percentage of luminescence normalized to DMSO-treated cells. The data are representative of two independent experiments. (B) Vero cells were treated as described in panel A and VEEV-TC83luc (MOI 5 or 10) was used for the infection. At 8 hpi, Promega’s BrightGlo Luciferase Assay was performed according to the manufacturer’s protocol. The graph represents percent luminescence compared to DMSO. To calculate EC₅₀ values, Vero cells were pre-treated for two hours with 1:2 serial dilutions of KPT-185 (C), KPT-301 (D), KPT-335 (E), or KPT-350 (F), then infected with VEEV-TC83luc (MOI 1) for one hour, cells post-treated with inhibitors, and the BrightGlo Luciferase Assay performed at 16 hpi. Biological triplicates of each concentration were averaged then presented as a percentage of luminescence compared to 1% DMSO-treated cells. The data are representative of two independent experiments.</p

Interferon stimulated genes are induced in the presence of SINE compounds.

<p>(A) MEFs were pre-treated with DMSO or KPT-185 (2.5 μM) for two hours, infected with VEEV-TC83 (MOI 1), and post-treated following infection. Mock infected cells were processed alongside as controls. RNA lysates were prepared at 16 hpi. qRT-PCR was performed using TaqMan Gene Expression Assays for IFIT1, IFIT2, IFNβ, and OASL1. 18S rRNA was used as the endogenous control. *p-value < 0.05. (B) MEFs were pre-treated as described in (A). After pre-treatment, 0 or 250 IU interferon-β was added, and RNA lysates were prepared four hours later. qRT-PCR was performed as described in (A).</p

Capsid mutations confer some resistance to SINE treatment.

<p>Vero cells were pretreated with DMSO (1%) or KPT-185 (2.5 μM) for two hours then infected with TC83-Wt, TC83-T41I, TC83-K64E, or TC83-K64M at a MOI of 1 for one hour. Cells were washed with 1X PBS and media containing either DMSO or KPT-185 added. Supernatants were collected 16 hpi, and titers determined by plaque assay using BHK-21 cells. Graphs represent biological triplicates. *p-value ≤ 0.05 (compared to KPT-185 treated cells infected with TC83-Wt).</p

SINE compounds altered capsid localization and reduced viral titer when used as a post-treatment only.

<p>(A) Vero cells were infected with VEEV-TC83 (MOI 1) for one hour. Media without drugs was added after infection. Four hours later, cells were post-treated with 2.5 μM of KPT-185, KPT-301, or DMSO (0.1%) for four hours. At 8 hpi, cells were fixed and probed for capsid (red) and DAPI stained (blue). The scale bar represents 50 μm, with each image captured at the same resolution. (B) Fn/c values were calculated to quantitate capsid localization. Treatment with the three active compounds, KPT-185, KPT-335, and KPT-350 had statistically significant accumulations of nuclear fluorescence, as comparable with Leptomycin B treatment. *** p-value ≤ 0.0001 (compared to DMSO treated cells). N is greater than 50 cells. (C) Vero cells treated as in (A), collected at 8 hpi, and plaque assays performed using Vero cells. Graphs represent biological triplicates and each experiment was performed twice. *** p-value ≤0.0001 (compared to DMSO treated cells).</p

Sequence analysis of VEEV capsid ORF.

<p>Sequence analysis of VEEV capsid ORF.</p

SINE compounds reduced released viral RNA and capsid.

<p>(A) Vero cells were pre-treated for two hours with DMSO (1%), Leptomycin B (45 nM), or KPT-185 (2.5 μM) prior to infection with VEEV-TC83 (MOI 1). Cells were post-treated after infection as well. At 4 and 8 hpi, supernatants were collected and extracellular viral RNA extracted and analyzed by q-RT-PCR. Graphs represent biological triplicates and each experiment was performed twice. Genomic copies were normalized as a percentage of the DMSO control (left panel) or shown without normalization (right panel). (B) Vero cells were treated as described above, and total intracellular RNA was extracted from lysed cells and analyzed by q-RT-PCR. Genomic copies were normalized as a percentage of the DMSO control (left panel) or shown without normalization (right panel). (C) Vero cells were treated and infected as described in panel A. At 22 hpi, supernatants (Sup) and whole cell lysates (WCL) were collected. Supernatants were purified using a sucrose gradient. Purified supernatants and WCL were assayed using western blotting and probed for capsid and actin as a loading control. M = Mock; D = DMSO. Right panel: Volume densities of supernatant and WCL capsid from the western blot were normalized to actin.</p

SINE compounds reduced VEEV-TC83, TrD, but not TC83_Cm titers.

<p>(A) Vero cells were treated for two hours prior to infection with DMSO (0.5%), 45 nM Leptomycin B, or 2.5 μM of KPT-185, KPT-301, KPT-335, or KPT-350. After an hour infection with VEEV-TC83 (MOI 1), the cells were post-treated with compounds. At 16 hpi, supernatants were collected and plaque assays performed using Vero cells. Graphs represent biological triplicates and each experiment was performed twice. (B) Vero cells were treated as described above and infected with WT TC83 or TC83_Cm (MOI 1). (C) Vero cells were treated as described above and infected with VEEV-TrD (MOI 1). (D) Vero cells were treated as described above and infected with VEEV-TC83 (MOI 1), with supernatants collected at 8, 16, and 24 hpi for plaque assays using Vero cells. Graphs represent biological triplicates. *p-value ≤ 0.05 and ** p-value ≤ 0.01 (compared to DMSO treated cells at the corresponding time point).</p

Treatment with SINE compounds altered capsid localization in VEEV-TC83 infected cells.

<p>(A) Vero cells were pre-treated with KPT-185 (2.5 μM), KPT-301 (2.5 μM), Leptomycin B (45 nM), or DMSO (0.1%) for two hours prior to infection with VEEV-TC83 or VEEV-TC83_Cm at a multiplicity of infection (MOI) of 1. Cells were post-treated after infection as well. Mock cells were left untreated and uninfected. At 16 hours post-infection (hpi), cells were fixed and probed for capsid (red) and DAPI stained (blue). Data are representative of at least three separate images per treatment group. The scale bar represents 50 μm, with each image captured at the same resolution. (B) Nuclear to cytoplasmic fluorescence ratios (Fn/c) were calculated to quantitate capsid localization. ***p-value ≤ 0.0001 (compared to DMSO treated cells). N is greater than 72 cells. (C) Fn/c values were calculated to quantitate capsid localization, as described in (B). N is greater than 63 cells.</p

Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection

<div><p>Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase protein array (RPPA) to define changes in signaling cascades after <i>in vitro</i> infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.</p></div