Role of the envelope glycoproteins in the infection cycle of tomato spotted wilt virus

Tomato spotted wilt virus (TSWV) forms the type member of the genus Tospovirus , which today harbors more than twelve different species. TSWV is able to infect an enormous variety of different plants, to which it often causes devastating effects, resulting in severe economical losses. Among the plant viruses, TSWV and the other tospoviruses form a distinct group. Taxonomically, they surprisingly do not belong to a plant virus family, but to a virus family which further consists of animal-infecting viruses, the Bunyaviridae . Consequently, they harbor features that are more common to animal-infecting viruses than to plant viruses. The most eye-catching animal-infecting virus-like feature of the tospoviruses is their envelope, in which two viral surface glycoproteins are embedded, denoted G1 and G2. These surface glycoproteins are designed for interaction with receptors, an important step in the infection of animals, but useless in the infection of plants. The plant-infecting tospoviruses are transmitted by thrips, in which they also replicate, and for the entry and circulation of the virus through this insect the glycoproteins are essential. This ensures their continues presence despite their lack of function during the infection of plants. The structure and function of the TSWV glycoproteins during infection in plants and insects form the subject of this thesis, of which the contents will be summarized in the next paragraphs, and also visually represented in Fig. 1. First develop the tools. Although the TSWV glycoproteins do not have a crucial function in the plant, they do play an essential role in the formation of virus particles. At the onset of these studies, however, no clear view of this morphogenesis process was available, so it was the obvious first goal to unravel this process for TSWV in plants. In the past, virus associated structures observed during the infection of whole plants were reported, but thirty five years of observations had not resulted in a clear model of the particle morphogenesis. Useful antibodies against the separate TSWV proteins had not been available, and most importantly, a system in which a synchronous infection could be investigated lacked, so that interpretation of the chrology of the events in the morphogenesis had been difficult. Chapter 2 of this thesis describes the development of a protoplast infection system for TSWV, which enabled the study of a synchronous TSWV infection in plant cells. Using newly produced antibodies against the viral glycoproteins together with antibodies against the nucleoprotein, it could be shown that a full, synchronous, TSWV infection is achieved, by the PEG-mediated inoculation of freshly isolated Nicotiana rustica protoplasts with freshly (and quickly) isolated TSWV particles. Similar inoculation of Vigna unguiculata protoplasts did not result in a full infection, since the production of enveloped particles was hampered due to low expression of the viral glycoproteins. to reveal the essence of TSWV particle making. Using the system, based on N. rustica protoplasts, the different virus associated structures could be assigned a chronological position in the morphogenesis process. This view of the chronology of the process revealed that the so-called paired parrallel membranes and doubly enveloped particles form essential intermediates in the process, which precede the accumulation of singly enveloped particles in the lumen of ER membranes. Using specific antibodies against the TSWV structural proteins, as well as antibodies against plant cell organels, the model could be completed ( Chapter 3 ). TSWV structural components, nucleocapsids as well as glycoproteins, accumulate at Golgi membranes, which are consequently modified to form the paired parrallel membranes. Doubly enveloped particles are formed by the so-called "wrapping" of these viral glycoprotein containing membranes around nucleocapsid cores, a process unique among plant viruses. The subsequent step is the fusion of these doubly enveloped particles with each other and specifically with ER membranes. This results in the formation of singly enveloped particles that accumulate within the ER. and then look what causes all this. After the complete model of the morphogenesis became available, the next step was to investigate what (molecular) features of especially the glycoproteins regulate the process. An important observation in the morphogenesis process is the apparent accumulation of viral glycoproteins in the Golgi system. This may, analogous to other enveloped viruses, be caused by the specific targeting of the glycoproteins to this organel due to a retention signal. The trafficking and retention behavior of TSWV glycoproteins was investigated in mammalian cells, the results of which are described in Chapter 4 . TSWV G1 and G2 accumulate in the Golgi system when expression together, which indeed implicates that at least one of the proteins must harbor a Golgi retention signal. Separate expression of G1 and G2 revealed that the retention signal is present in G2. G1 on its own is transport incompetent, but this can be rescued by the co-expression with G2, which suggests that G1 is dependent on, and interacts with G2 during transport and retention. These molecular features, identified in mammalian cells, are most probably also functional in plant cells, causing the observed accumulation of glycoproteins in the plant Golgi system during infection. The TSWV glycoproteins thereby show their crucial role in directing the particle morphogenesis process. and how these particles interact with thrips. Once the particles are formed and accumulated inside ER membranes, they await the uptake by the thrips vector to be transferred to another plant. Earlier research has shown that between this uptake and the release of virus there is replication and circulation of TSWV in the thrips. However, nothing was known about the molecular interactions between TSWV proteins and proteins of the thrips during this process. In Chapter 5 an overlay blot technique was used to investigate the possible binding of TSWV structural proteins with thrips proteins, that could be potential receptors involved in entry or circulation of TSWV. A 94 kDa thrips protein was identified, displaying specific binding to TSWV G2 protein. This 94 kDa protein was found in known vectors of TSWV, and also in a non-vector thrips species, albeit it not in the larval stages of the latter. Although a receptor is anticipated in the gut of the vectoring insects, the 94 kDa protein is not found there. It is however present in all other parts of the thrips body, suggesting that it may have a role during the replication and circulation of the virus. Fig. 1 Diagram of TSWV infection in a plant cell, indicating the scope of the experimental chapters of this thesis