Impact on the Endoplasmic Reticulum and Golgi Apparatus of Turnip Mosaic Virus Infection

Abstract

The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K 2 . TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K 2 fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K 2 vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K 2 vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.

Turnip mosaic virus Moves Systemically through Both Phloem and Xylem as Membrane-Associated Complexes

“…1B, right, bottom dashed rectangle) showed that 6K 2 :GFP-tagged structures are approximately 0.5-mm vesicles. This is consistent with what is observed in live epidermal cells (Grangeon et al, 2012), which indicates that tissue fixation did not produce any apparent artifactual structures.…”

“…These intracellular vesicles are the building blocks of viral factories, which contain viral replication complexes. In the case of TuMV, these same vesicles also are the vehicles for the intracellular and intercellular movement of vRNA (Grangeon et al, 2012. It thus appears that the TuMV RNP complexes found in vascular conducting tubes are, in fact, membraneassociated viral replication complexes.…”

“…Moreover, the dsRNA recognized by the monoclonal antibody is not likely to be encapsidated vRNA, since the immunohistolocalization protocol does not involve protease treatment, and gold particles are associated with vesicles and not viral particles in immunogold electron microscopy images following incubation with the dsRNA monoclonal antibody. The vesicles in sieve elements and xylem vessels are reminiscent to those found in the cytoplasm of TuMV (Grangeon et al, 2012) and are very similar to other plant and mammalian virus-infected cells (for review, see Miller and Krijnse-Locker, 2008;Laliberté and Sanfaçon, 2010). These intracellular vesicles are the building blocks of viral factories, which contain viral replication complexes.…”

“…Hence, our study indicates that membrane-associated TuMV replication complexes are involved in the systemic movement of the virus. TuMV factories in N. benthamiana epidermal cells are characterized by the presence of numerous 100-nm 6K 2 -tagged vesicles containing replication complexes, many of them aggregating to form a large irregularly shaped globe-like structure near the nucleus (Grangeon et al, 2012). Different types of cells have different structures and functions.…”

“…TuMV remodels cellular membranes into viral factories, which are intracellular compartments involved in viral replication and movement. These compartments take the form of vesicles of approximately 100 nm in diameter originating from the endoplasmic reticulum (Grangeon et al, 2012). These vesicles contain viral RNA (vRNA) and viral and host proteins involved in vRNA replication Dufresne et al, 2008;Huang et al, 2010;Grangeon et al, 2012).…”