Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3

Abstract

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.

Keywords: TFAP2A; Venezuelan equine encephalitis virus; eIF2S2; host-proteome; mass spectrometry; non-structural protein 3; small molecule inhibitors; viral proteome.

Figure 1

Mass spectrometry identified putative host interactors with VEEV nsP3 and potential antivirals. Then, 293T cells were transfected with a pcDNA 3.1⁽⁺⁾ empty control plasmid or a pCAGGS plasmid expressing N-terminus HA-tagged nsP3 of the ZPC738 VEEV strain. Next, 24 hpt, lysates were subjected to immunoprecipitation with anti-HA tag antibody or IgG3 isotype control antibody. Expression and immunoprecipitation of HA-nsP3 was confirmed by Western blot (A) and six independent immunoprecipitated samples were subjected to Fusion Orbitrap mass spectrometry (B), as described in Materials and Methods. Western blot and immunoprecipitations are representative of six independent experiments (n = 6). Mass spectra were fitted against the NCBI reference sequence AAB02516 or P27282 for the non-structural polyprotein of VEEV. (C) Schematic of data analysis by Ingenuity Pathway Analysis. Host proteins identified by mass spectrometry runs were analyzed for abundancy and identification of canonical pathways, networks, and inhibitors. (D) A list of small molecule inhibitors were identified using IPA for efficacy studies.

Figure 2

Efficacy of inhibitors against VEEV TC-83 in U-87MG astrocytes. (A) U-87MG cells were treated with small molecule inhibitors at varying concentrations (μM). Cell viability was measured at 24 hpt and calculated versus the DMSO vehicle control, as described in Materials and Methods. The dotted line represents the 90% cut-off point. (B) Schematic of experimental setup. (C) U-87MG cells were treated with a single, nontoxic concentration of inhibitor or 0.1% DMSO for 2 h. Cells were infected with TC-83 at MOI of 0.1 in triplicate for 1 h. Conditioned media containing inhibitor was replaced after removal of the virus. At 24 hpi, viral supernatants were collected and evaluated by plaque assay, as described in Materials and Methods. Graph represents data from two independent experiments performed in triplicate (n = 6). * p < 0.0332, **** p < 0.0001, ns, not significant.

Figure 2

Efficacy of inhibitors against VEEV TC-83 in U-87MG astrocytes. (A) U-87MG cells were treated with small molecule inhibitors at varying concentrations (μM). Cell viability was measured at 24 hpt and calculated versus the DMSO vehicle control, as described in Materials and Methods. The dotted line represents the 90% cut-off point. (B) Schematic of experimental setup. (C) U-87MG cells were treated with a single, nontoxic concentration of inhibitor or 0.1% DMSO for 2 h. Cells were infected with TC-83 at MOI of 0.1 in triplicate for 1 h. Conditioned media containing inhibitor was replaced after removal of the virus. At 24 hpi, viral supernatants were collected and evaluated by plaque assay, as described in Materials and Methods. Graph represents data from two independent experiments performed in triplicate (n = 6). * p < 0.0332, **** p < 0.0001, ns, not significant.

Figure 3

Tomatidine, citalopram HBr, and Z-VEID-FMK can inhibit TC-83 replication in multiple cell types. SVGp12 and HMC3 cells were treated with two concentrations of tomatidine, citalopram HBr, and Z-VEID-FMK. (A,B) Cell viability was measured at 24 hpt and calculated versus the DMSO vehicle control, as described in Materials and Methods. The dotted line represents the 90% cut-off point. (C,D) Cells were treated and infected in triplicate, as previously described (MOI 0.1), and viral supernatants were collected and evaluated by plaque assay, as described in Materials and Methods. Graph represents data from two independent experiments performed in triplicate (n = 6). * p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p < 0.0001, ns, not significant.

Figure 4

Selectivity indexes of tomatidine, citalopram HBr, and Z-VEID-FMK against VEEV TC-83. U-87MG cells were treated with varying micromolar concentrations of tomatidine, citalopram HBr, and Z-VEID-FMK. (A) cell viability was measured at 24 hpt and calculated versus the DMSO vehicle control, as described in Materials and Methods. The dotted line represents the 90% cut-off point. (B) Schematic of infection scheme (MOI 0.1). (C) Viral supernatants were collected at 24 hpi and evaluated by plaque assay, as described in Materials and Methods. Graph represents data from two independent experiments performed in triplicate (n = 6).

Figure 5

Time-course and post-exposure efficacy against VEEV TC-83. (A) U-87MG cells were pretreated for 2 h with nontoxic concentrations of tomatidine, citalopram HBr, and Z-VEID-FMK, subsequently infected with TC-83 at MOI 0.1 for 1 h, and conditioned media were replaced after the removal of viral inoculum. Viral supernatants and intracellular lysates were collected at 6, 12, and 24 hpi. (D) U-87MG cells were infected with TC-83 for 1 h at MOI 0.1. Nontoxic concentrations of tomatidine, citalopram HBr, and Z-VEID-FMK were added after the 1-h infection (0 h) or at 2, 4, 6 h post-infection. A 2-h pretreatment group with all drugs were included as a control. At 24 hpi, viral supernatants and intracellular lysates were collected. (B,E) Viral supernatants were evaluated by plaque assay, as described in Materials and Methods. (C,F) qRT-PCR to measure positive strand expression levels (nsP3) and RT-PCR to measure expression levels of negative strand was performed, as described in Materials and Methods. Graph represents data obtained from two independent experiments performed in triplicate (n = 6). ** p < 0.0021, *** p < 0.0002, **** p < 0.0001, ns, not significant.

Figure 6

Tomatidine, citalopram HBr, and Z-VEID-FMK display efficacy against the wild-type VEEV Trinidad donkey strain in a cell-type independent manner. (A) Schematic of infection scheme. Cells were pretreated with two nontoxic concentrations of tomatidine, citalopram HBr, or Z-VEID-FMK for 2 h, subsequently infected with VEEV TrD at MOI 0.1 (B,D,E) or MOI 1 (C) for 1 h, and conditioned media were replaced after the removal of viral inoculum. Viral supernatants were evaluated by plaque assay, as described in Materials and Methods. (B,C) Efficacy in U-87MG cells at MOI 0.1 and 1, respectively. (D) Efficacy in SVGp12 cells. (E) Efficacy in HMC3 cells. Graphs are representative of two independent experiments performed in technical triplicates (n = 6). **** p < 0.0001.

Figure 7

Tomatidine, citalopram HBr, and Z-VEID-FMK display efficacy against EEEV in a cell-type independent manner. (A) Schematic of infection scheme. Cells were pretreated with two nontoxic concentrations of tomatidine, citalopram HBr, or Z-VEID-FMK for 2 h, subsequently infected with EEEV at MOI 0.1 for 1 h, and conditioned media were replaced after the removal of viral inoculum. Viral supernatants were evaluated by plaque assay, as described in Materials and Methods. (B) Efficacy in U-87MG cells. (C) Efficacy in HMC3 cells. (D) Efficacy in SVGp12 cells. Graphs are representative of two independent experiments performed in technical triplicates (n = 6). **** p < 0.0001.

Figure 8

EIF2S2 supports a pro-viral role for VEEV TC-83 replication and VEEV nsP3 colocalizes with TFAP2A. (A) 293T cells were transfected with 25 or 50 nM siRNA targeting TFAP2A or eIF2S2, with an off-target negative control siRNA, DharmaFECT only treatment, or mock-transfection treatment and incubated for 72 h. Cell viability was measured, as described in Materials and Methods and calculated versus mock-transfected control. Dotted line represents the 90% cut-off point. (B) SiRNA knockdown and infection scheme. Then, 293T cells were transfected with 25 or 50 nM siRNA targeting TFAP2A or eIF2S2 and incubated for 72 h. Cells were infected with TC-83 for 1 h at MOI 0.1 and at 24 hpi, viral supernatants and cellular lysates were obtained, as described in Materials and Methods. Graph is representative of two independent experiments performed in triplicate (n = 6). Cellular lysates to confirm host protein knockdown were probed using Western blot for TFAP2A (C) or eIF2S2 (D) as described in Materials and Methods. Signal was quantified and normalized to actin loading control, calculated as fold-change vs. mock-transfected control group. Western blot and graphical data are representative of two independent experiments (n = 2). (E) Viral supernatants of siRNA transfected cells were evaluated by plaque assay, as described in Materials and Methods. Graph is representative of two independent experiments performed in triplicate (n = 6). Images are representative of two independent experiments. * p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p < 0.0001, and ns, not significant.

Figure 9

EIF2S2 is important for VEEV genomic RNA but not subgenomic RNA translation. Furthermore, 293 T cells were transfected with 50 nM eIF2S2 or off-target negative siRNA for 72 h or treated with 10 µM Tomatidine for 2 h. TC-83 genomic RNA containing a mutated stop codon was transfected, as described in Materials and Methods for 24 h. Cellular lysates were probed for eIF2S2 siRNA knockdown (A,B), nsP2 as an indicator of genomic translation (A,C), as described in Materials and Methods. Signals were quantified and normalized to the actin loading control and fold-change was calculated versus the mock-transfected cells (B) or the untreated and non-siRNA transfected WT-Stop control (C). Western blot and graphical data are representative of two independent experiments (n = 2). SiRNA transfected or tomatidine treated 293 T cells were infected in triplicate with a nLUC-TaV expressing TC-83 virus at MOI 0.1 for 24 h, as described in Materials and Methods. (D) Cellular lysates were obtained and quantified for luciferase activity and normalized to total protein by Bradford assay for measurement of subgenomic RNA translation, as described in Materials and Methods. Graph is representative of two independent experiments performed in triplicate (n = 6). * p < 0.0332, ** p < 0.0021, *** p < 0.0002, **** p < 0.0001, ns, not significant.