Fig 1.
Impact of HsNMT isozymes on infectious CVB3 production.
HAP1 cells (wt, NMT1KO, NMT2KO) were infected with CVB3 at an MOI of 1 and infectious virus titers were measured 7 h p.i. Each bar represents the mean ± SD, n = 3; * p < 0.05 using the two-tailed Student´s t test; ns: not significant.
Fig 2.
DDD85646 dramatically reduces CVB3 infectious titers.
(A) Structural formula of the myristoylation inhibitors DDD85646 and (B) 2-HMA. (C) Effect of DDD85646 and (D) 2-HMA on HeLa cell viability (XTT assay) at the indicated concentrations and times. Each data point represents the mean ± SD, n = 9. (E) HeLa cells were infected with CVB3 at an MOI of 1 and DDD85646 was added 1 h p.i. at the indicated concentrations, with DMSO used as solvent control. Cell lysates prepared 7 h p.i. were used to determine virus yield by end point titration and the data are expressed as the 50% tissue culture infective dose (TCID50) per ml. Each bar represents the mean ± SD, n = 3. (F) HeLa cells were infected with CVB3 at an MOI of 1, treated 1 h p.i. with the specified concentrations of 2-HMA and with DMSO as solvent control, lysed and virus titer quantified as before. Each bar represents the mean ± SD, n = 3.
Table 1.
Antiviral activity of DDD85646 against various picornaviruses.
Fig 3.
DDD85646 massively reduces metabolic incorporation of the Alk-12 myristate analogue into VP0.
(A) Work flow for detection of Alk-12 labeled viral proteins with click reagents. HeLa cells (DDD85646- or DMSO-treated) are infected with CVB3 at MOI of 10. The myristic acid analogue Alk-12 is added 4 h p.i. and incubation continued for 3 h; during this time Alk-12 becomes transferred to viral substrate proteins by metabolic incorporation. Cells are lysed and Alk-12-bearing proteins are ligated to the fluorescent reporter 5-TAMRA-azide by click chemistry, separated by SDS-PAGE and visualized by in-gel fluorescence. (B) DDD85646 at 5 μM blocks incorporation of Alk-12 into CVB3 VP0. The gel subjected to in-gel fluorescence recording was subsequently stained with InstantBlue to visualize total proteins for verifying equal loading.
Fig 4.
DDD85646 does not affect CVB3 replication.
(A) Effect of the time-of-drug addition of DDD85646 (5 μM) on the infectious titer of progeny CVB3. The well-known inhibitor of viral replication, guanidine hydrochloride (GuHCl) was used as a control at 2 mM and DMSO served as solvent control. Each bar represents the mean ± SD, n = 3. (B) Viral RNA replication of RLuc-CVB3 in the absence (DMSO control) and presence of 5 μM DDD85646 as measured by the luciferase activity assay. GuHCl (2 mM) was used as a control inhibitor. Each bar represents the mean ± SD, n = 3. (C) The viral RNA level during a CVB3 single cycle infection was measured by RT-qPCR in the absence (DMSO control) and presence of 5 μM DDD85646 at the indicated time points and normalized to the GAPDH mRNA level. Each data point represents the mean ± SD, n = 2.
Fig 5.
DDD85646 efficiently suppresses CVB3 cell-to-cell transmission.
(A) HeLa cells were infected with CBV3-eGFP at an MOI of 0.1 in absence (DMSO control) or presence of 5 μM DDD85646 and observed under the fluorescence microscope 5 h and 24 h p.i. (B) Cells remaining intact after the 24 h multi-cycle infection with CVB3-eGFP were subsequently challenged with wt CVB3 in complete absence of the NMT inhibitor and visualized for a CPE 24 h later. Scale bar 5 μm.
Fig 6.
NMT inhibition by DDD85646 results in impaired CVB3 VP0 processing and drastically diminished specific infectivity.
(A) Western blot analysis of VP1, VP3, VP0 and VP2 expression 6 h p.i. in lysates of Alk-12 metabolically labeled HeLa cells infected with CVB3 (MOI of 10) and treated with 5 μM DDD85646 or solvent control (DMSO). Tubulin served as a loading control. (B) HeLa cells were infected with CVB3 at an MOI of 5 in absence (DMSO control) or presence of DDD85646 (5 μM). Cell lysates were prepared at 7 h p.i. and the number of encapsidated (SuperNuclease-protected) genomes determined by RT-qPCR. Each bar represents the mean ± SD, n = 3. (C) The yield of infectious progeny virus in lysates prepared in (B) was determined by endpoint titration. The derived TCID50/ml values for the drug- and solvent-treated samples were multiplied by 0.7 to obtain PFU/ml. (D) Specific infectivity calculated from the data in (B) and (C) as the number of PFU per 1010 viral RNA genomes of progeny CVB3 propagated in the presence of DDD85646 or the solvent control (DMSO). Each bar represents the mean ± SD, n = 3.
Fig 7.
Growth of CVB3 in presence of DDD85646 results in accumulation of provirions.
(A) Effect of DDD85646 on small (early) viral assembly intermediates. HeLa cells were infected with CVB3 in absence (DMSO control) or presence of the drug (5 μM) and lysates prepared 7 h p.i. were loaded onto a 5–25% (w/v) sucrose density gradient. Odd fractions taken from top to bottom were analyzed for 5S protomers and 14S pentamers by Western blotting using VP1 and VP0/VP2-specific antibodies. The resuspended pellet was similarly analyzed for the presence of large viral structures which sedimented to the bottom of the tube. Cartoon representations of the small assembly intermediates are displayed above the corresponding regions in the blot. The sucrose density is expressed as g/cm3. Note that images of Western blots from two different gels prepared under identical conditions were stitched together (indicated by a short-dashed line) for better appreciation of the results (B) Effect of DDD8546 on the assembly of large (late) viral assembly intermediates. HeLa cells were infected with CVB3 in absence (DMSO control) or presence of the drug (5 μM) and viral assemblies ≥14S present in lysates prepared 7 h p.i. were initially pelleted through a 30% (w/v) sucrose cushion. The respective resuspended material was loaded onto a 10–30% (w/v) sucrose density gradient for separation of 75S and 150S particles. Only 1/10 of the control sample was used to compensate the higher virus production in absence of DDD85646. Fractions were collected from top to bottom and capsid proteins visualized by Western blotting as described before. Cartoon representations of the assembly intermediates are displayed above the corresponding regions in the blot. The sucrose density is expressed as g/cm3. Images of Western blots from two different gels prepared under identical conditions were stitched together (indicated by a short-dashed line) for better appreciation of the results (C) Gradient fractions corresponding to 75S empty procapsids and 150S full particles (virions and provirions) were pooled separately, pelleted, and recovered particles were visualized by negative-stain transmission electron microscopy. Scale bar 100 nm.
Fig 8.
Inhibition of myristoylation by DDD85646 does not perturb CVB3 2C and VP1 colocalization.
HeLa cells were infected with CVB3 (MOI of 5) in presence of 5 μM DDD85646 or DMSO (solvent control) and fixed and permeabilized with 0.5% saponin at 5 h p.i. Nuclei were visualized with DAPI (blue color). The location of VP1 was determined with a mouse monoclonal anti-enterovirus VP1 primary antibody and Alexa Fluor 488-conjugated secondary antibody (green color). 2C was probed in the same cells with an anti-CVB3 2C rabbit antibody and Alexa 555-conjugated secondary antibody (red color). Images were acquired by confocal immunofluorescence microscopy. Yellow regions in the merged images indicate colocalization. Scale bar 20 μm.
Fig 9.
Ultrastructural changes of DDD85646- or control-treated HeLa cells infected with CVB3.
(A) TEM image of a noninfected HeLa cell treated with 0.1% DMSO (solvent control). N, nucleus; Nm, nuclear membrane; M, mitochondria; ER, endoplasmic reticulum; MV, microvilli; Ch, chromatin. (B) TEM image of a noninfected HeLa cell treated with 5 μM DDD85646. (C) TEM image of a HeLa cell infected with CVB3 at an MOI of 10 in the presence of 0.1% DMSO (solvent control). The observed ultrastructural changes are typical for infection by this virus. DMV, double-membrane vesicle; MMV, multimembrane vesicle; Vb, virus bleb (an irregular virus-related aggregate); Ar, virus paracrystalline array. The black arrows indicate discontinuations of the nuclear envelope. Insets C1 to C3 are higher magnifications of the boxed regions. (D) TEM image of a HeLa cell infected with CVB3 at an MOI of 10 in the presence of 5 μM DDD85646. Virus blebs are present but no paracrystalline arrays are formed. Insets D1 and D2 are higher magnifications of the boxed regions.