| Viruses are intracellular pathogens that use the host cell to produce new infectious progeny. For all positive-strand RNA viruses that have been investigated so far, viral replication takes place in cytoplasmic virus-induced membrane structures. For turnip mosaic virus (TuMV), the generation of vesicles likely associated with the replication complex depends on the synthesis of the viral protein 6K. To further characterize the vesicles formed during TuMV infection, Nicotiana benthamiana plants were agroinfiltrated with a TuMV infectious clone expressing 6K protein fused to GFP. Using confocal microscopy, cytoplasmic aggregates were observed, corresponding to the 6KGFP-induced vesicles which house the viral replication complexes (VRCs). Intracellular movement of these vesicles was visualized by time-lapse imaging. Vesicle trafficking was inhibited when plants were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. The absence of movement had severe effects on viral accumulation. Viral vesicles also aligned with actin filaments. These results indicate that microfilaments are necessary for VRC trafficking which is important for virus infectivity.;The biogenesis of viral vesicles was investigated by infecting cells with two recombinant TuMVs producing 6KGFP or 6KmCherry-labelled vesicles. Individual vesicle within a cell contained unique protein products derived from each recombinant demonstrating the origin of a vesicle from a single viral genome. Green and red sectoring was also observed, meaning that vesicles could fuse together. The presence of the eukaryotic translation factors eIF(iso)4E, PABP and eEF1A enclosed in VRC was demonstrated previously by our group. These data combined to a single-genome origin suggest that viral translation occur within these structures. The same host factors were also found to co-localize with the active replicating sites along with viral proteins VPg-Pro, RdRp and CI using immunofluorescence labelling in infected protoplasts. These data bring accumulating evidence for the possible coupling of viral translation and replication. |
