Nanotrap particles can capture and enrich NP from virally infected cells.

<p>A) One ml of cytoplasmic extract (CE) at 2.6 μg/ml obtained from RVFV infected Vero lysates were incubated with 100 μl of NT45, NT53, and NT69. No Nanotrap particle sample (-NT) was processed in parallel. After 30 minutes, the (+)NT samples were centrifuged. The unbound material (S) from the spin was saved and 10 μl was processed in parallel. The bound material (P) was resuspended in blue lysis buffer and boiled for 10 minutes. The samples were centrifuged at maximum speed and the supernatants were then loaded onto a NuPage 4–12% Bis-Tris gel. Samples were subsequently analyzed by western blot for NP. B) One ml of CE was serially diluted in 50mM Tris-HCl from 15 μg/ml to 0.75 μg/ml and incubated with 100 μl of NT45 for 30 minutes. No Nanotrap particle samples (-NT) were processed in parallel. The control sample is CE at 770 μg/ml (10 μl volume). The samples were processed as in panel A.</p

Non-virion associated NP can be detected in viral supernatants.

<p>Vivaspin 20 centrifugal concentrators with a 300,000 Da MWCO were used to filter viral supernatants harvested from Vero cells infected at an MOI 1 with MP12. Five milliliters of viral supernatant was added to the concentrators and centrifuged at 1400 rpm for 7 minutes until approximately 500 μl remained on the top portion of the concentrator. A) The top (T) and bottom (B) fractions, as well as control sample (C) containing the original sample before processing were analyzed by western blot using antibodies against NP (EC22 antibody). C, T, and B lysates were undiluted (neat). Cytoplasmic extract (CE) control is 7.7 μg/ml of RVFV infected Vero cell lysates. B) Plaque assays were performed with both fractions and the control sample. C) Densitometry analysis was performed to determine the NP band densities in the T and B fractions. The percent of total NP was determined by dividing the top or bottom portion's band density by the total NP band density (top and bottom portions added together) and multiplying by 100. D) NP band density per pfu values were calculated by dividing the NP band densities by the total pfu values.</p

Nanotrap particles can protect NP from degradation at increased temperatures and times.

<p>A and B) Cytoplasmic extracts from RVFV infected cells were diluted in 100% sheep serum for a final concentration of 7.7 μg/ml. C and D) Viral supernatant was diluted in 100% goat serum for a final titer concentration of 1E+06 pfu/ml. One milliliter of sample was incubated with NT45 for 24 to 120 hours at either ambient temperature (A and C) or at 37^°C (B and D). No Nanotrap particle (-NT) and control cytoplasmic extract (CE) samples at 7.7μg/ml or 0.77 μg/ml (B only) at 10 μl volumes were processed in parallel. Viral supernatants without Nanotrap particles containing samples were analyzed on a separate gel (E). After the incubation time, the (+)NT samples were centrifuged and washed once with 0.25M sodium thiocyanate and twice with diH₂O. Presence of NP was analyzed by western blot using antibodies against NP.</p

Schematic of antigen capture with Nanotrap particles.

<p>Nanotrap particles are incubated with samples for 30 minutes at room temperature, centrifuged, and unbound material is removed. The pellet is resuspended in lysis buffer and boiled for 10 minutes. The sample is then centrifuged and unbound supernatant is loaded onto an SDS PAGE gel.</p

Nanotrap particles can capture RVFV NP.

<p>A) Recombinant histidine-tagged NP (His-NP) at a starting concentration of 0.4 mg/ml and a volume of 100 μl was incubated with 75 μl of NT45, NT46, NT53, NT55, NT69, or NT71 for 30 minutes at ambient temperature. After 30 minutes, the (+)NT samples were centrifuged. Both bound (P) and 10uL of unbound (S) material was resuspended in blue lysis buffer and boiled for 10 minutes. The samples were centrifuged at maximum speed and the supernatants were then analyzed for presence of NP protein by western blot using antibodies directed against the histidine tag. No Nanotrap particle samples (-NT) at a 10 μl volume of 0.4 mg/ml His-NP were processed in parallel. B) Purified NP (obtained from BEI Resources) at 2 μg in a volume of 100 μl was incubated with 75 μl of NT45, NT46, NT53, NT55, NT69, or NT71 for 30 minutes at an ambient temperature. A control—NT sample (10 μl volume) was processed in parallel. Samples were processed as describe in panel A. After electrophoresis, NP was visualized by Commassie blue staining. C) His-NP (obtained from Immune Technology) at 1 μg/ml and a volume of one milliliter was incubated with 100 μl of NT45, NT53, and NT69. A control—NT sample (10 μl volume) was processed in parallel. The control sample is 100 μg/ml NP (volume of 10 μl). The samples were processed as in panel A and analyzed by western blotting for NP by using antibodies against NP. D) Viral supernatants at 1E+06 pfu/ml and a volume of 1 ml were incubated with 100 μl NT45, NT46, NT53, NT55, and NT69 for 30 minutes at an ambient temperature. Control—NT samples (10 μl volumes) at 1E+07pfu/ml and 1E+06 pfu/ml were processed in parallel. The samples were processed as in panel A and analyzed by western blot for NP.</p

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-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

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

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