Efficacy and Mechanism of Action of Low Dose Emetine against Human Cytomegalovirus

Abstract

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 μM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Anti-HCMV activity of emetine and synergy with GCV.

A) Cells were infected with pp28-luciferase HCMV Towne and treated with indicated concentrations of emetine or GCV. Luciferase activity was measured at 72 hpi. Data represent mean ± SE of triplicate determinations from a representative of three independent experiments. B) Cells were treated with the indicated concentrations of emetine and cell viability was determined after 72 h. Data represent mean values ± SE of triplicate determinations from a representative of three independent experiments. C) Inhibition of HCMV protein expression by emetine (75 nM) or GCV (5 μM) at 72 hpi. Cells were infected with HCMV Towne and treated with emetine. Western blot of viral proteins IE1/2, UL44 and pp65 was performed at 72 hpi. D) Cells were infected with pp28-luciferase HCMV Towne, and treated with combination of GCV and emetine. The antiviral activity was evaluated by luciferase assay. Data represent mean values ± the SE of triplicate determinations from a representative of three independent experiments.

Fig 2. Emetine is an early inhibitor of HCMV replication.

A) Emetine does not inhibit HCMV entry. Cells were treated with emetine (75 nM), GCV (10 μM), and CpG 2006 (10 μM) 24 h prior to infection. Cells were infected with HCMV and treated with compounds for 90 min. Immunofluorescence staining was performed with mouse monoclonal anti-pp65 antibody. The fluorescence of rhodamine anti-mouse IgG and DAPI was visualized and merged using a Nikon Eclipse E-800 fluorescence microscope. B) Emetine has an early activity against HCMV. Cells were infected with HCMV Towne, and compounds were added at 0, 6, 12, 24, 36, and 48 hpi (Add on). Culture supernatants (10%) were collected at 72 hpi for a plaque assay after 14 days. C) Cells were infected with HCMV Towne and treated with compounds immediately after virus adsorption. Compounds were removed at 0, 6, 12, 24, 36, and 48 hpi (Removal). Culture supernatants were collected at 72 hpi for titration by plaque assay. Data represent mean ± SE of triplicate determinations from a representative of two independent experiments.

Fig 3. Emetine achieves high tissue concentrations and is efficacious against MCMV replication.

A)In vivo pharmacokinetics of emetine. Plasma, liver, lung and spleen samples were collected at the indicated time points after single oral administration of emetine at 0.1 mg/kg in male BALB/c mice. Concentrations were measured using UPLC-MS/MS methods. For plasma, the concentrations observed were mostly below the level limit of quantification. B) Quantitative real-time PCR of viral gB was performed on DNA extracted from blood at day 14 post infection. Plaque assay was performed from salivary glands C) or liver D) collected at day 14 post infection. Emetine was administered orally starting 24 hpi or 72 hpi at 0.1 or 1.0 mg/kg every 3 days. GCV dose was 10 mg/kg/dose administered intraperitoneally twice daily.

Fig 4. The anti-HCMV activity of emetine is reduced in low-density HFFs.

A) Cells were seeded at indicated densities in a 96-well plate, infected with pp28-luciferase HCMV Towne (MOI = 1) and treated with emetine (75 nM) or GCV (5 μM). Luciferase activity was measured at 72 hpi and normalized to the activity of infected untreated cells. Data represent mean ± SE of triplicate determinations from a representative of two independent experiments. B) Same cell lysates as in A were collected for Western blotting at 72 hpi and expression of viral proteins UL44 and pp65 was determined. C) Supernatants from each well as in A were used to infect cells (1 million cells in a 96-well plate) in the corresponding well and luciferase activity was measured at 72 hpi. D) Cells were seeded into 12-well plates at 0.5 and 2 million cells/plate, infected with 100 PFU/well of HCMV Towne or MCMV, and treated with emetine (75 nM) or GCV (5 μM). After 10 days (for HCMV) or 3 days (for MCMV) plaques were stained with crystal violet and the number of plaques enumerated. Data shown are average of 2 wells (±SD) for a representative experiment from two different experiments.

Fig 5. Emetine induces an interaction between MDM2 and RPS14 in infected high-density cells.

A) Emetine abrogates infection-mediated overexpression of RPS14 at 72 hpi. Cells were seeded at 0.5 or 2 million cells/plate, infected with Towne HCMV and treated with emetine (75 nM) or GCV (5μM). Lysates were collected at 24 or 72 hpi for Western blotting. B) HFFs were seeded at 1 or 2 million cells/plate and treated with emetine or GCV. Lysates were collected at 72 hpi for Western blotting. C) Cells were seeded at 0.5 or 2 million/plate in 100 mm dishes, infected with Towne HCMV followed by treatment with emetine (75 nM) or GCV (5μM) for 24h. MG132 (10 μM) was added after 12 h. At 24 hpi, lysates were collected and subjected to IP with anti-MDM2 followed by immunoblotting with anti-RPS14 antibody or D) In reverse reaction, IP was performed with anti-RPS14 followed by immunoblotting with anti-MDM2 antibody. Densitometry analysis was performed to normalize immunoprecipitated protein to its input level. E) A model showing the interaction of MDM2 and RPS14 in high-density but not in low-density cells.

Fig 6. Emetine induces nuclear translocation of RPS14 in infected high-density cells at 24 hpi followed by cytoplasmic relocalization.

A) Cells were seeded at 2 million/4-well chamber slide, infected and treated with either emetine (75 nM) or GCV (5μM) for 24 h. Cells were stained with IE1/2 (Alexa 555:Red) for evidence of infection and RPS14 (FITC: Green) and nuclear DAPI. Stained slides were subjected to confocal microscopy and colocalization was studied and quantified using NIS elements software (Nikon). B) The same procedure was repeated at 72 hpi. The images shown are a representative of two independent experiments. Percent nuclear localization is represented as Mean ± SD from two different fields of at least 40 cells per condition.

Fig 7. Nuclear localization of RPS14 persists during infection and emetine treatment in low-density cells.

A) Cells were seeded at 0.5 million/4-well chamber slide, infected and treated with either emetine (75 nM) or GCV (5μM) for 24 h. Cells were stained with IE1/2 (Alexa 555:Red) for evidence of infection and RPS14 (FITC: Green) and nuclear DAPI. Stained slides were subjected to confocal microscopy and nuclear localization was quantified using NIS elements software (Nikon). B) The same procedure was repeated at 72 hpi. The images shown are a representative of two independent experiments. Percent nuclear localization is represented as mean ± SD of at least 25 cells per condition from two different fields in the slide.

Fig 8. RPS14 is ubiquitinated and degraded in infected high-density cells.

A) Cells were seeded at 0.5 or 2 million/plate in 100 mm dishes, infected with HCMV Towne followed by treatment with emetine (75 nM) or GCV (5μM) for 72h. MG132 (10 μM) was added 12 h before harvest. Lysates were collected at 72 hpi and subjected to IP with anti-Ubiquitin antibody followed by immunoblotting with anti-RPS14 antibody or IP with anti-RPS14 antibody followed by immunoblotting with anti-Ubiquitin antibody. The mono- and poly- ubiquitinated forms of RPS14 are depicted as (Ub)-RPS14 and n(Ub)-RPS14, respectively. B) HCMV-infected cells (left: high-density right: low-density) were treated with emetine or GCV. At 24 hpi cycloheximide (CHX, 100 μg/mL) was added. Cells were harvested at the indicated time intervals up to 8 h following CHX for SDS-PAGE analysis.

Fig 9. Emetine disrupts HCMV mediated MDM2-p53 complex.

A) MDM2 and p53 expression increases with emetine treatment. Cells were seeded at 0.5 or 2 million cells/plate, infected with HCMV Towne followed by treatment with emetine (75 nM) or GCV (5 μM). Cell lysates were harvested at 24 or 72 hpi for Western blotting. B) Cells were seeded at 2 million cells/plate, infected with HCMV Towne and treated with emetine (75 nM) or GCV (5 μM). RNA was harvested and qRT-PCR was performed for p21. Data represent mean ±SE of triplicate determinations from a representative of three independent experiments C) HFFs were seeded at 1 or 2 million cells/plate and treated with emetine (75 nM) or GCV (5 μM). Cell lysates were harvested at 72 hpi for Western blotting. D) HFFs were seeded at either 0.5 or 2 million/plate in 100 mm dishes, infected with HCMV Towne followed by treatment with emetine (75 nM) or GCV (5μM) for 24h. MG132 (10 μM) was added after 12 h. At 24 hpi, lysates were collected and subjected to IP with anti-MDM2 antibody followed by immunoblotting with anti-p53 antibody or E) IP with anti-p53 antibody followed by immunoblotting with anti-MDM2 antibody. F) A model depicting the mechanism by which emetine disrupts HCMV mediated MDM2-p53 interaction in high-density cells, but not in cycling low-density cells.

Fig 10. Emetine loses its anti-HCMV activity in RPS14 knockdown cells.

A) RPS14 knockdown HFFs (sh-RPS14) were generated from lentiviral system and expression of RPS14 in the knockdown cells was compared to TRC control cells by Western blotting. B) sh-RPS14 and TRC control cells were seeded at 2 million/plate, infected and treated with emetine (75 nM) or GCV (5μM) for 72h. Cell viability was determined after 72 h. C) Luciferase activity was measured in cell lysates. D) sh-RPS14 and TRC control cells were seeded at 2 million/plate, in a 12-well plate and infected 24 hours later at 100 PFU/well. After 90 min virus was aspirated, and DMEM containing 4% fetal bovine serum (FBS) with 0.5% carboxymethyl-cellulose, were added with the compounds at indicated concentrations into triplicate wells. Plaques were counted after incubation at 37°C for 8 days. E) Same cell lysates as in B were used to determine HCMV pp65, UL44 and IE1/2 expression. F) sh-RPS14 and TRC-control cells were infected and treated with emetine (75 nM) for 24h. MG132 (10 μM) was added after 12 h. At 24 hpi, lysates were collected and subjected to IP with anti-p53 antibody followed by immunoblotting with anti-MDM2 antibody.

Fig 11. Model of HCMV inhibition by emetine.

In high-density infected cells (A) emetine induces (1) nuclear translocation of RPS14 (2) followed by RPS14 binding to MDM2 (3 & 4) resulting in disruption of the interaction between MDM2-p53 (6) and MDM2- viral IE2 (5 & 7), and by RPS14 ubiquitination and degradation (8). In low-density infected cells (B) although emetine induces (1) nuclear translocation of RPS14 (2), it is unable to interact with MDM2 (4) which is already bound to p53 to facilitate virus replication (3).