The Cowpea mosaic virus movement protein

For systemic infection of a host plant, viruses multiply in the initially infected cell and spread to the neighbouring cells through plasmodesmata (cell-to-cell movement), to eventually reach the vascular system and use the phloem to spread to other plant parts (long-distance movement). To achieve cell-to-cell transport through plasmodesmata, these complex pores in the plant cell wall must be modulated to allow viral passage. Two major types of cell-to-cell transport have been described, movement of the viral genome in a non-encapsidated form, as exemplified by Tobacco mosaic virus (TMV), and "tubule-guided" movement of mature virus particles (virions), exemplified by Cowpea mosaic virus (CPMV). In both mechanisms one or more virally encoded movement proteins (MP) play an essential role in the targeting of infectious entities from the site of replication to the plasmodesmata, as well as in the subsequent modification of and transport through the modified pores. However, it is generally recognised that intercellular movement is a concerted effort of not only viral factors but also host factors, the knowledge of the latter being very scarce at the moment.With CPMV, the MP polymerises within the plasmodesmal pore into a transport tubule, through which mature virions then are delivered into the neighbouring uninfected cell. Identical tubules are also formed in single plant protoplasts that are infected with CPMV or transfected with the MP gene alone, hence, in the absence of cell wall and plasmodesmata.At the onset of the research presented in this thesis, no information about host proteins interacting with the CPMV MP was available. Such interactions were to be expected, for instance during the process of transport (targeting) of the MP from its site of synthesis to the periphery of the infected cell, the polymerisation process at the plasma membrane, and the structural modification of the plasmodesma. Thus, the research described in this thesis focused on the functioning of the CPMV MP with special emphasis on its interactions with virion proteins and host proteins.For initial studies on these interactions the property of the CPMV MP to assemble into tubules on single cell protoplasts was exploited in Chapter 2. Thus it was shown that virus particles residing in the tubule contain a single deviant species of the small coat protein (S CP) that is larger than the two forms of S CP (S-s and S-f) which are consistently found in virus present in the cytoplasm of infected cells. The nature of the deviation is not known, but the exclusive presence of this deviant S CP in virions that are being transported suggests that the S CP is in some way involved in cell-to-cell movement.Identification of host proteins in isolated tubule fractions by electrophoretic analysis was not successful, but a directed search for potential host proteins by Western blot analysis using specific antibodies indicated the presence of pectin methylesterase (PME) in the plasma membrane surrounding the tubule (Chapter 2). This protein has previously been implicated in cell-to-cell movement of other plant viruses, i.e. TMV, Cauliflower mosaic virus and Turnip vein clearing virus . The PME enzyme is involved in cell wall turnover and affects cell wall rigidity by modulating pH and ion balance. Such cell wall dynamics could be a necessity for the modification of the plasmodesmal pore to enable the insertion of a viral transport tubule.The interaction between the MP and virion proteins was further investigated in Chapter 3. Protein overlay assays and ELISA showed that the MP binds only to its homologous virions and that it is the large (L) coat protein which is involved in this binding. Considering also the deviation found in the S CP of virions within the transport tubules, it is conceivable that both CPs play a crucial but different role in the cell-to-cell movement of CPMV. A C-terminal deletion in MP, which in planta results in a mutant virus defective in cell-to-cell movement and producing tubules devoid of particles, also resulted in the abolishment of L CP binding, thus validating the in vitro binding approaches.The ability of the CPMV MP to bind nucleic acid and rNTP was analysed in Chapter 4. It is shown that MP binds rGTP but no other rNTPs, and by site-directed mutagenesis the GTP binding site was located within a sequence motif conserved among the MPs of tobamo- and comoviruses. The non-GTP-binding mutant MP exhibited disturbed intracellular targeting and tubule formation, suggesting that GTP binding may play a significant role in targeted transport and multimerization of the MP. It was also shown that the MP is capable of binding both ss-RNA and DNA, but not ds nucleic acids.The studies on possible interactions between CPMV MP and host (plasma membrane) proteins were extended in Chapter 5. To identify potential MP-binding host proteins, purified MP was used as a probe in overlay assays and affinity column chromatography to assess plasma membrane proteins for their affinity to the MP. In the blot overlay assays, candidate MP-binding proteins with apparent sizes of 34, 30 and 28 kDa were detected. Further analysis of the cowpea plasma membrane fraction using affinity chromatography also revealed a limited number of eight MP-binding proteins including again a 30 kDa protein band. Sequencing of the 30 kDa protein band revealed that it actually represented a mixture of two protein species, i.e. an aquaporin and a vacuolar-type ATPase. A possible role of these host proteins in viral MP functioning is discussed in Chapter 5.Finally, in the General Discussion (Chapter 6) the results obtained in this thesis research are discussed and integrated in a speculative model for the functioning of the CPMV MP, accommodating the different observed interactions with virion and host proteins