Structure, morphogenesis and function of tubular structures induced by cowpea mosaic virus

During systemic plant infection, viruses move from the initially infected cells through plasmodesmata to neighbouring cells. Different mechanisms have been proposed for this cell-to-cell movement. Cowpea mosaic virus (CPMV) employs one of the major movement mechanisms, i.e. tubule-guided transport of virions, and this mechanism has been the subject of this thesis. The tubule-guided movement mechanism involves the assembly of movement protein (MP) into tubular structures within the plasmodesmal channel of infected cells, which pave the way for translocation of mature virus particles. These transport tubules are also induced on isolated plant cells (protoplasts) in the absence of cell walls and plasmodesmata. Employing this protoplast system, the structure, morphogenesis and function of CPMV tubules was studied.By mutational analysis of RNA-2 of CPMV (Chapter 2) it was established that mutations in the coding region of the overlapping 48 kDa/58 kDa proteins, but not the capsid proteins, resulted in abolishment of tubule formation in protoplasts. Deletion of the capsid proteins resulted in the formation of tubules without virions. As the 58 kDa protein contains the entire 48 kDa sequence, mutations made in the overlapping coding region affect the function of both proteins. To establish the involvement of each protein in tubule formation, antisera specific to the unique 10 kDa N-terminus of the 58 kDa protein were made by using synthetic peptides. Although the antisera reacted to purified 58 kDa protein in immunoblots, they failed to react in immunocytochemical experiments. Alternatively, the 58 kDa gene alone was transiently expressed in protoplasts. In these protoplasts no tubular structures were formed and the 58 kDa protein apparently localized to the nucleus. As production of the 48 kDa protein in protoplasts was previously shown to result in tubule formation it was concluded that this protein constitutes the actual viral movement protein (MP). The possible function and significance of the 58 kDa protein are discussed in Chapter 3.Having established that the 48 kDa protein is the viral MP responsible for tubule induction, the possible role of host proteins in this process was investigated following two different approaches. First, the expression of the MP gene in a heterologous (insect) cell system was studied (Chapter 4). As movement of plant viruses occurs through plasmodesmata, intercellular channels unique to plant cells, it was speculated that plant specific host factors could play a role in targetting and assembly of the tubules.However, upon production of the MP in insect cells, tubule formation occurred in a fashion similar to that in plant protoplasts. This led to the conclusion that if host proteins were involved in this mechanism, these should be of a conserved (among plant and animal) nature (Chapter 4). The second approach involved the isolation and subsequent biochemical analysis of tubules from infected protoplasts. This showed that the MP was (apart from the coat proteins) the sole major component of the movement tubules and host proteins were not obviously present in these structures (Chapter 5).Tubule-guided movement of virions appears to be an important mechanism used by a large variety of plant viruses. For two other viruses which are genetically unrelated to CPMV, i.e. brome mosaic virus and alfalfa mosaic virus, evidence for this mechanism was obtained during the course of this PhD research. These two representatives of the Bromoviridae are genetically more closely related to tobacco mosaic virus (TMV) which supposedly does not move using a tubule-guided mechanism, but moves as an RNA-MP complex through plasmodesmata. The fact that the MP of these viruses also forms tubules in protoplasts indicates that tubule formation may be a more general ability of plant virus MPs (Chapter 6). This hypothesis is discussed and corroborated by literature data and additional experimental evidence in Chapter 7.