Mining a Kröhnke Pyridine Library for Anti-Arenavirus Activity

Several arenaviruses cause hemorrhagic fever (HF) disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus is a neglected human pathogen of clinical significance. There are no licensed arenavirus vaccines, and current antiarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel therapeutics to combat human pathogenic arenaviruses, a task that will be facilitated by the identification of compounds with antiarenaviral activity that could serve as probes to identify arenavirus–host interactions suitable for targeting, as well as lead compounds to develop future antiarenaviral drugs. Screening of a combinatorial library of Krönhke pyridines identified compound KP-146 [(5-(5-(2,3-dihydrobenzo­[b]­[1,4] dioxin-6-yl)-4′-methoxy-[1,1′-biphenyl]-3-yl)­thiophene-2-carboxamide] as having strong anti-lymphocytic choriomeningitis virus (LCMV) activity in cultured cells. KP-146 did not inhibit LCMV cell entry but rather interfered with the activity of the LCMV ribonucleoprotein (vRNP) responsible for directing virus RNA replication and gene transcription, as well as with the budding process mediated by the LCMV matrix Z protein. LCMV variants with increased resistance to KP-146 did not emerge after serial passages in the presence of KP-146. Our findings support the consideration of Kröhnke pyridine scaffold as a valuable source to identify compounds that could serve as tools to dissect arenavirus–host interactions, as well as lead candidate structures to develop antiarenaviral drugs

Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na⁺/K⁺ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses

<div><p>Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na⁺/K⁺ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.</p></div

Effect of knock-down of genes identified LC-MS/MS analysis on LCMV multiplication.

<p><b>(A)</b> Effect of siRNA-mediated knockdown expression of genes identified by proteomics analysis. A549 cells (1,000 cells/well) in a 384-well plate were reverse transfected with siRNA pools targeting each indicated gene. At 72 h post-transfection, cells were infected (MOI = 0.05) with rLCMV/ZsG. At 48 h pi, cells were fixed and stained with DAPI. ZsGreen and DAPI signals were measured by a fluorescence plate reader. ZsGreen signal was normalized to DAPI signal. Normalized values from cells transfected with the control (NC, black bar) non-specific siRNA was set to 100%. A panel of 154 siRNAs including NC siRNA was evaluated three independent times. Results from three independent experiments were sorted by mean values and assigned to three graphs with SD. Each graph includes identical NC results (black bar) for reference. <b>(B)</b> Reduction of protein expression of ATP1A1 and PHB by siRNA-mediated gene knockdown. A549 cells (3.0 x 10⁴ cells/well) were reverse transfected in a 24-well plate with siRNA pools against either ATP1A1 or PHB or with NC siRNA. At 72 h post-transfection, total cell lysate was prepared, and expression of ATP1A1 and PHB in cell lysates were determined by western blots. <b>(C)</b> Effect of siRNA-mediated kd of ATP1A1 on production of infectious LCMV progeny. A549 cells (1.5 x 10⁴ cells/well; 48-well plate) were reverse-transfected with siRNAs against ATP1A1 or with NC siRNA. 72 h later, cells were infected (MOI = 0.01) with rLCMV/ZsG. At 24 h and 48 h pi, TCSs were collected. At 48 h pi, cells were fixed and ZsGreen expression examined by fluorescence microscopy (i). Bar, 200 μm. Virus titers in TCSs were determined by IFFA (ii). Data represent means ± SD of results from three independent experiments. LoD, limit of detection.</p

Identified host-cell proteins with spectral counts at least 2-fold higher in NP pull down samples than eGFP samples.

Identified host-cell proteins with spectral counts at least 2-fold higher in NP pull down samples than eGFP samples.</p

Endogenous BST-2 expression levels have a very limited impact on LCMV propagation <i>in vitro</i>.

<p><b>A.</b> Cell surface expression of BST-2 in HeLa-TKD and HeLa-pLKO cells. Cells were fixed and analyzed by FACS using either a control antibody (Ab) or anti-BST-2 conjugated to PE. <b>B.</b> Growth kinetics of LCMV in HeLa-pLKO and HeLa–TKD cells. Cells were infected with LCMV (moi = 0.01). At 24 and 48 hrs p.i, virus titers in TCS were determined by plaque assay (n = 3; 2 independent experiments). <b>C.</b> Propagation of LCMV in HeLa-pLKO and HeLa-TKD cells. Cells were infected with LCMV (moi = 0.1). At 24 and 48 h p.i., cells were fixed with 4% PFA and after permeabilization stained with a MAb to LCMV NP, followed with a second antibody conjugated to FITC. Nuclei were stained with DAPI. Mock infected cells were used as control. <b>D.</b>Similar Z protein expression levels in HeLa-TKD and HeLa-pLKO cells at 16 h p.i. Cells were infected (moi = 0.1) with rLCMV/Z-FLAG and at 16 h p.i. expression levels of Z protein determined by WB using a MAb to FLAG. Levels of actin were used as loading control. <b>E-F.</b> Production of virion particles by LCMV-infected HeLa-TKD and HeLa-pLKO cells. Cells were infected with rLCMV/Z-FLAG (moi = 0.1) and at 24 hrs p.i. virion particles present in TCS were recovered by ultracentrifugation, and cell lysates prepared. Levels of Z protein present in virion particle preps and cell lysates were determined by WB using an Ab to FLAG (<b>E</b>), and signals quantified with LAS3000 (Fuji Film) (<b>F</b>). The ratio of virus/cell Z protein levels in HeLa-pLKO was set to 1.0 (n = 3; 3 independent experiments). <b>G.</b> Growth kinetics of VSV in HeLa-pLKO and HeLa–TKD cells. Cells were infected with VSV (moi = 0.01). At 8, 18 and 24 h p.i, virus titers in TCS were determined by plaque assay (n = 3; 2 independent experiments). Data correspond to mean + SD. Asterisks (*) denote statistical significance (<i>P</i> < 0.05).</p

Synergistic antiviral effect of ouabain and rocaglamide on rLCMV/eGFP multiplication.

<p>A549 cells seeded (2.0 x 10⁴ cells/well) in 96-well plates and cultured overnight were treated with combinations of ouabain and Roc-A at indicated concentrations for 2 h and then infected (MOI = 0.01) with rLCMV/eGFP. Compounds were present throughout the end of experiment. At 48 h pi, cells were fixed and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader. eGFP signal was normalized to DAPI signal, and the normalized data were used to analyze synergistic effect by MacSynergy II software. Data represent % synergy (% inhibition over the expected [additive effect]) at the 95% confidence interval from five independent experiments.</p

LC-MS/MS analysis of NP-binding proteins.

<p><b>(A)</b> Flow chart of the experimental approach to identify NP-interacting host-cell proteins in LCMV-infected cells. A549 cells prepared in six 15-cm dishes (total of 1.0 x 10⁸ cells) were infected (MOI = 0.1) with either rLCMV/Strep-NP or r3LCMV/eGFP. At 48 h pi, total cell lysates were prepared, and NP- or eGFP-interacting proteins were pulled down (PD) using Streptactin-coated sepharose resin. Protein complexes bound to the resin were eluted using 2.5 mM of desthiobiotin. Eluates were precipitated using TCA followed by trypsin digestion. Tryptic peptides were subjected to LC-MS/MS analysis. <b>(B)</b> Detection of proteins present in PD samples. Protein complexes present in PD samples were separated by SDS-PAGE and visualized by SYPRO staining. Some protein bands present only in the Strep-NP PD sample are indicated by asterisks. <b>(C)</b> Venn diagram of the NP- and eGFP-interacting proteins identified by LC-MS/MS analysis. <b>(D, E)</b> Gene Onthology (GO) analysis of the NP-interacting proteins identified by LC-MS/MS. Bioinformatic analysis by PANTHER was performed showing the number of genes of identified NP-interacting proteins classified by biological process <b>(D)</b> and protein class <b>(E)</b>.</p

BST-2 contains LCMV within the splenic marginal zone.

<p><b>A-B.</b> Representative confocal images from naïve, d3 WT, and d3 BST-2 KO mice depict the splenic distribution of LCMV Cl-13 (green; A) or CD169+ metallophillic macrophages (green; B) in relation to BST-2 expression (red) (n = 4 mice per group; 2 independent experiments). <b>C.</b> Representative confocal images captured in the spleens of WT vs. BST-2 KO mice at day 3 post-infection show the distribution of LCMV (red) in relation to the white pulp (WP), marginal zone (MZ), and red pulp (RP). Laminin staining is shown in green to delineate these anatomical regions. <b>D.</b> Quantification of the LCMV staining pattern shown in panel C (n = 8 mice per group; 2 independent experiments). Data are represented as mean ± SD. Asterisks denote statistical significance (*P < 0.05).</p

BST-2 deficiency impedes control of acute and persistent strains of LCMV.

<p><b>A.</b> Serum viral titers were quantified by plaque assay in WT vs. BST-2 KO mice infected with LCMV Cl-13 (n = 5 mice per group; 2 independent experiments). <b>B.</b> Brain viral loads were quantified by Q-PCR [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007172#ppat.1007172.ref048" target="_blank">48</a>] in WT vs. BST-2 KO mice 237 days following infection with LCMV Cl-13 (n = 3–4 mice per group; 2 independent experiments). <b>C.</b> Serum viral titers were quantified by plaque assay in WT vs. BST-2 KO mice 5 days following infection with LCMV Arm (n = 3–4 mice per group; 2 independent experiments). All data in this figure are represented as mean ± SD. Asterisks denote statistical significance (*P < 0.05).</p

BST-2 deficiency changes the splenic distribution and proliferative capacity of antiviral CD8+ T cells.

<p><b>A.</b> Representative confocal images were captured at day 4 post-infection in the spleens of WT and BST-2 KO mice seeded with mOrange+ P14 cells (green). The splenic distribution of P14 cells in relation to LCMV Cl-13 (red) is shown. The white dotted line demarcates the border between the white pulp (WP) and red pulp (RP). <b>B.</b> The bar graph shows quantification of the white vs. red pulp P14 percentage in WT vs. BST-2 KO mice (n = 4 mice per group; 2 independent experiments). Data are represented as mean ± SD. Asterisks denote statistical significance (*P < 0.05). <b>C.</b> The representative histogram depicts the dilution of CFSE in P14 cells from WT (blue) vs. BST-2 KO (red) mice at day 3 post-infection. <b>D.</b> The bar graph shows quantification of the CFSE dilution data in panel C (n = 4 mice per group; 2 independent experiments). Graphed are the percentage of P14 cells that divided more than 5 times. Data are represented as mean ± SD. Asterisks denote statistical significance (*P ≤ 0.05).</p