The cellular biology of turnip mosaic virus

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Genome replication of positive-strand RNA viruses is closely associated with membranes of the infected cell. For instance, turnip mosaic virus (TuMV) infection induces in the infected cells the formation of elaborate membranous organelle-like structures that sustain viral RNA synthesis and virus intercellular movement. The TuMV genome consists of a single RNA molecule of ~10 000 nucleotides of positive polarity. This RNA codes for a single polyprotein that is processed by three viral proteinases to yield eleven mature proteins. One protein, namely the 6K2 protein, is responsible for membrane recruitment. Two 6K2-induced structures are observed in infected cells: a large perinuclear globular structure that leads to the production of several small motile peripheral vesicles. These vesicles contain viral RNA, viral replication proteins and host factors, indicating that 6K2 vesicles harbour viral replication complexes. 6K2 vesicles are also found in association with plasmodesmata and can move directly into adjoining cells. Symplasmic movement of TuMV is thus achieved as a membrane-associated virus replication complex associated with 6K2. The intercellular movement of these vesicles depends on the secretory pathway and on myosin motors. My research objectives for the next 5 years will be to further elucidate the architecture of these membrane rearrangements and to identify some of the molecular events that lead to these changes. More precisely, my research team will use transmission electron microscopy (TEM) and tomography to get a 3-dimensional view of some of the cellular remodelling that are observed in infected cells and in vascular tissues, particularly in xylem vessels. We will also investigate the role of the tonoplast in virion assembly. My group will also map the molecular determinants of 6K2 that are involved in the formation of the vesicles. Preliminary experiments have indicated that the 6K2 protein has a tryptophan-based endoplasmic reticulum export signal that interacts with COPII coatomer components. We will confirm the involvement of these host proteins as well as the SNARE-like vesicle-associated protein (VAP) AtPva11, AtPva12 , and AtPva22 in 6K2 vesicle biogenesis. Finally, we will screen for host factors that are part of 6K2-associated viral replication complexes using a proteomic approach and validate their involvement in TuMV infection.