Tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum- and Golgi-derived pleomorphic membrane structures in plant cells

Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts. When separately expressed, Gc was solely observed in the endoplasmic reticulum (ER), whereas Gn was found both within the ER and Golgi membranes. Upon co-expression, both glycoproteins were found at ER-export sites and ultimately at the Golgi complex, confirming the ability of Gn to rescue Gc from the ER, possibly due to heterodimerization. Interestingly, both Gc and Gn were shown to induce the deformation of ER and Golgi membranes, respectively, also observed upon co-expression of the two glycoproteins. The behaviour of both glycoproteins within the plant cell and the phenomenon of membrane deformation are discussed in light of the natural process of viral infectio

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 cell

Requirements for ER-Arrest and Sequential Exit to the Golgi of Tomato Spotted Wilt Virus Glycoproteins

The envelope glycoproteins Gn and Gc are major determinants in the assembly of Tomato spotted wilt virus (TSWV) particles at the Golgi complex. In this article, the ER-arrest of singly expressed Gc and the transport of both glycoproteins to the Golgi upon co-expression have been analyzed. While preliminary results suggest that the arrest of Gc at the ER (endoplasmic reticulum) did not appear to result from improper folding, transient expression of chimeric Gc, in which the transmembrane domain (TMD) and/or cytoplasmic tail (CT) were swapped for those from Gn, showed that the TMD of Gn was sufficient to allow ER-exit and transport to the Golgi. Expression of both glycoproteins in the presence of overexpressed Sar1p-specific guanosine nucleotide exchange factor Sec 12p, resulted in ER-retention demonstrating that the viral glycoproteins are transported to the Golgi in a COPII (coat protein II)-dependent manner. Inhibition of ER-Golgi transport by brefeldin A (BFA) had a similar effect on the localization of Gn. However, inhibition of ER (endoplasmic reticulum) to Golgi transport of co-expressed Gc and Gn by overexpression of Sec 12p or by BFA revealed distinct localization patterns, i.e. diffuse ER localization versus concentration at specific spot

Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta

Recently, the Tomato Spotted Wilt Virus (TSWV) Gn and Gc glycoproteins were shown to induce the formation of (pseudo-) circular and pleomorphic membrane structures upon transient expression in plant cells. Furthermore, when singly expressed, Gc retains in the ER, while Gn is able to further migrate to the Golgi. Upon co-expression, Gn rescues Gc and co-migrates to the Golgi complex. Here, we have studied the behavior of the glycoproteins in the presence of the viral nucleocapsid (N) protein and in vivo analyzed the occurrence of protein¿protein interactions by fluorescence life time imaging microscopy (FLIM). The analysis demonstrated that N co-localizes and interacts with both glycoproteins, with a preference for Gn. Additionally, it is shown that N causes a dramatic change in the distribution of Gc within the ER, from reticular to punctate spots. The observations are discussed in the context of the virus particle formation during the infection process

The Cytosolic Nucleoprotein of the Plant-Infecting Bunyavirus Tomato Spotted Wilt Recruits Endoplasmic Reticulum–Resident Proteins to Endoplasmic Reticulum Export Sites

In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N). During maturation of the plant-infecting bunyavirus Tomato spotted wilt, Gc localizes at endoplasmic reticulum (ER) membranes and becomes ER export competent only upon coexpression with Gn. In the presence of cytosolic N, Gc remains arrested in the ER but changes its distribution from reticular into punctate spots. Here, we show that these areas correspond to ER export sites (ERESs), distinct ER domains where glycoprotein cargo concentrates prior to coat protein II vesicle–mediated transport to the Golgi. Gc concentration at ERES is mediated by an interaction between its cytoplasmic tail (CT) and N. Interestingly, an ER-resident calnexin provided with Gc-CT was similarly recruited to ERES when coexpressed with N. Furthermore, disruption of actin filaments caused the appearance of a larger amount of smaller ERES loaded with N-Gc complexes, suggesting that glycoprotein cargo concentration acts as a trigger for de novo synthesis of ERE