A model for the biogenesis of turnip mosaic virus replication factories.

Nicotiana benthamiana plants were agroinfiltrated with an infectious clone of the Turnip mosaic virus (TuMV) that was engineered to tag replication vesicles with either GFP or mCherry fluorescent proteins. Punctuate vesicle structures were observed in the cytoplasm of infected cells corresponding to viral replication factories. The vesicles were highly motile and co-aligned with the microfilaments. Utilization of latrunculin B, an inhibitor of microfilament polymerization, reduced accumulation of the virus, suggesting that microfilaments are necessary during infection. To investigate biogenesis of the vesicles, leaves were infected simultaneously with two recombinant TuMV infectious clones, one that labeled vesicles in red and one that labeled them in green. We observed cell with green only and red only vesicles indicating a single viral genome origin. In some cases, vesicles exhibited sectors of green, red and yellow fluorescence were also observed, demonstrating that fusion among individual vesicles is possible. Based on those results we propose a model for the biogenesis of viral factory, where viral translation and replication are tightly coupled within virus-induced vesicles.