Article Figures & Data
Figures
- FIG. 1.
PT and HK dramatically reduce picornavirus multiplication and increase cell viability. Viral titers of HRV2-infected HeLa cells (MOI = 20) in the presence or absence of indicated concentrations of PT (A) or HK plus 30 mM MgCl2 (B) were determined 24 h p.i. Results for one representative experiment of three experiments are shown. HeLa cells were infected with HRV2 (MOI = 50) or mock infected, and different concentrations of PT (C) or HK-MgCl2 (D) were added. At 24 h p.i., a CellTiter 96 AQueous nonradioactive cell proliferation assay was performed according to the manufacturer's protocol. Extinction at 492 nm reflects cell viability. The means ± standard deviations of quintuples obtained in one representative experiment of three performed are shown. (E) HeLa cells were infected with CVB3 or mengovirus (MOI = 10) and incubated with 10 μM PT or 75 μM HK-30 mM MgCl2, respectively. Virus production of infected cells was determined by TCID50 assay at 24 h p.i. The results for one of two representative experiments are shown. −, control.
- FIG. 2.
PT and HK abolish HRV2-triggered eIF4GI cleavage and affect viral polyprotein processing. (A) HRV2-infected HeLa cells (MOI = 20) were either mock treated or treated with 10 μM PT or 125 μM HK-30 mM MgCl2. After the indicated time points, protein extracts were collected and analyzed by Western blotting using an antibody specific for eIF4GI and its cleavage products. (B) HeLa cells were preincubated with 10 μM PT or 125 μM HK-30 mM MgCl2 for 30 min and infected with HRV2 (MOI = 50) in the presence of the substances. At 6 h p.i., newly synthesized proteins were labeled with [32S]Met/Cys for 1 hour. At the indicated time points, VP2 or VP2-containing precursor proteins were isolated by immunoprecipitation and analyzed by SDS-PAGE and autoradiography. Immunoprecipitation of mock-infected labeled cells did not show any signal. MW, molecular weight in thousands.
- FIG. 3.
PT and HK abrogate 3D and 3C processing of CVB3. HeLa cells infected with coxsackievirus B3 (MOI = 50) were incubated with 125 μM PDTC, 75 μM HK-30 mM MgCl2, or 10 μM PT in the presence or absence of 10 μM TPEN. At 5 h p.i., cells were starved half an hour prior to 1 h of labeling with [35S]Met/Cys. Harvested cell lysates were subjected to immunoprecipitation using either antibodies specific for 3D (left panel) or 3C (right panel) and then analyzed by SDS-PAGE and autoradiography. Results for a representative experiment are shown. −, TPEN absent; +, TPEN present.
- FIG. 4.
Zinc ions are responsible for the antiviral property of PT and HK. HeLa cells were infected with HRV2 (MOI = 20) (A), CVB3 (MOI = 10) (B), or mengovirus (MOI = 10) (C) and incubated with 10 μM EDTA or 10 μM of Zn-EDTA, Ca-EDTA, and Mg-EDTA, respectively. If indicated, 10 μM PT (A, B, and C), 125 μM HK-30 mM MgCl2 (A), or 75 μM HK-MgCl2 (B and C) was added at the time of infection. The virus titers in the supernatants of the infected cells were determined by a TCID50 assay at 24 h p.i. and shown as log TCID50/ml. Viral infection without addition of compounds is labeled as “cont.” The results for one representative experiment of three are shown.
- FIG. 5.
PDTC, PT, and HK cause import of extracellular 65Zn2+ levels in a dose-dependent manner. (A) HeLa cells in serum-free culture medium were incubated with 5 μM 65Zn2+ and 125 μM PDTC, 10 μM PT, or 125 μM HK-30 mM MgCl2. At the indicated time points, the cells were harvested and washed with a buffer containing EDTA, and the amount of intracellular 65Zn2+ was determined in a Packard Cobra II γ-counter. Statistical variations between different samples are determined to be a maximal ±10% (data not shown). The results for one of three experiments are shown. (B) HeLa cells in serum-free culture medium were incubated with 5 μM 65Zn2+ and various concentrations of PDTC (top panel), PT (middle panel), or HK-MgCl2 (lower panel) for 15 min. Then, the cells were collected and washed, and the amount of intracellular 65Zn2+ was determined as described above. The results for one of five experiments are shown.
- FIG. 6.
Zinc ionophores influence the labile Zn2+ pool. HeLa cells were loaded with 5 μM FluoZin-3 (A) or 1 μM RhodZin-3 (B) in PBS for 15 min at 37°C. After extensive washing and 1 h of de-esterification in medium, zinc uptake was induced by treatment with 125 μM PDTC, 10 μM PT, and 125 μM HK-30 mM MgCl2 in growth medium. If indicated, after 30 min of chemical treatment, ionophores were removed for 1 hour (PDTC + 1 h removal, PT + 1 h removal, HK + 1 h removal). Pictures were taken at the indicated time points by live fluorescence microscopy with 60× optics. Examples of images of three independent experiments were chosen.
