Cellular N-myristoyltransferases play a crucial picornavirus genus-specific role in viral assembly, virion maturation, and infectivity
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.