Towards understanding TSWW particle assembly: analysis of the intracellular behavior of the viral structural proteins

At the onset of the studies presented in this thesis, it was already known that the assembly of the enveloped particle of Tomato spotted wilt virus (TSWV; family Bunyaviridae) in the infected plant cell was featured by a number of interesting phenomena. This process involves enwrapment of the viral ribonucleoparticles (RNPs) by Golgi membranes. As a consequence, doubly enveloped virus particles (DEVs) are formed which, by fusion with each other and ER-derived membranes, give rise to large cytoplasmic vesicles that contain accumulating amounts of mature singly enveloped virus particles (SEVs), which are not secreted from the cell. Furthermore, since TSWV also replicates in its insect vector (thrips), viral assembly should be compatible with the membranous organelles and intracellular transport pathways present in both plant and insect cells. To gain more insight into the sequential steps that eventually lead to particle assembly, and on the longer term be able to identify the key factors involved in the observed differences in intracellular targeting between plant and insect cells, the behavior of the viral structural proteins, i.e. the nucleocapsid (N) protein and the two envelope glycoproteins Gc and Gn, was studied in plant cells. To this end a N. tabacum protoplast system was established that supported efficient transient expression of these proteins. By using fluorophore-fusions of these proteins, the interactions between one-another as well as their co-localization with specific elements of the endomembrane system and/or cytoskeleton could be monitored by confocal microscopy. In Chapter 2, the intracellular localization and behavior of the TSWV glycoproteins were studied. In the absence of Gn, Gc was found to be retained in the ER, whereas Gn was able to, by itself, target to the Golgi complex. When co-expressed, Gn was able to rescue Gc from the ER, and co-translocate to the Golgi complex. Occasionally both glycoproteins were also observed at ER export sites. Surprisingly, Gc and Gn were able to induce the formation of (pseudo-) circular/pleomorphic membrane structures that co-localized with ER or Golgi markers, respectively. In Chapter 3, the behavior of the glycoproteins was analyzed in the presence of the nucleocapsid protein N and FRET/FLIM was applied to the in vivo study of protein-protein interactions. Upon single expression, the N protein formed large cytoplasmic agglomerates due to homo-oligomerization as indicated by FRET/FLIM. N was also able to interact with both Gc and Gn at the ER and/or Golgi. Furthermore, a surprising concentration of Gc at specific areas of the ER was observed upon co-expression with N. The translocation of N and dependence on cytoskeleton elements was analyzed in Chapter 4. The formation of large cytoplasmic agglomerations of N was found to be dependent on active actin filaments, but independent of microtubules. However, the interaction of N with the glycoproteins was not affected by any of the cytoskeleton inhibitors tested, suggesting that microtubules and actin filaments were not required for the transport of N to the ER and/or Golgi. The specific change in the localization of the glycoprotein Gc within the ER by interaction with N was further investigated in Chapter 5 and it was shown that the two proteins co-localize at ER export sites. The interaction between N and the cytoplasmic tail of Gc was shown to be crucial for this localization since N was also able to induce the concentration at ER export sites of the ER-resident protein calnexin, upon exchange of its cytoplasmic tail by the one of Gc. Our results also suggested (some kind of) a connection between ER export sites and microtubules, since depolymerized a-tubulin was found to co-localize with the marker for these specific ER domains. The ER-retention of Gc upon single expression was further analyzed in Chapter 6. The characteristics of the transmembrane domain (TMD) of the glycoprotein were shown to be determinant for its ER-exit, since exchange by the TMD of Gn rendered its translocation to the Golgi complex. Furthermore, the TMD and CT of Gn were shown not to be sufficient for the interaction between the two glycoproteins, suggesting the importance of their luminal domain in this interaction. Using ER-to-Golgi transport inhibitors, viral glycoprotein transport to Golgi was demonstrated to occur via COPII vesicles and appeared to be sensitive to brefeldin A. In Chapter 7 the major findings of this PhD research were discussed in a broader perspective and combined in a model for TSWV particle assembly in plant cells