The structural phenotype of the movement proteins (MPs) of two representatives of the Bromoviridae, alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), was studied in protoplasts. Immunofluorescence microscopy showed that the MPs of these viruses, for which there has been no evidence of a tubule-guided mechanism, assemble into long tubular structures at the surface of the infected protoplast. Electron microscopy and immunogold analysis confirmed the presence of both MP and virus particles in the tubules induced by AMV and BMV. The significance of the tubule-forming properties of these viral MPs is discussed.

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On the surface of eowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M eDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wildtype virus.

Tubular structures involved in the cell-to-cell movement of cowpea mosaic virus (CPMV) were partially purified from infected cowpea protoplasts to identify the structural components. A relatively pure fraction could be obtained by differential centrifugation and this was analysed by PAGE and immunoblotting. Besides the movement protein (MP) and capsid proteins (CP) of CPMV, no other major infection-specific proteins could be detected, suggesting that host proteins are not a major structural component of the movement tubule.Intercellular movement of cowpea mosaic virus (CPMV) is achieved by transport of virions through specialized tubules that are assembled in modified plasmodesmata. It was previously shown in planta that the capsid protein (CP) and the 48 kDa movement protein (MP) of CPMV were located in these tubules (van Lent et al., 1990) and mutation or deletion of these proteins resulted in abolition of cell-to-cell movement (Wellink & van Kammen, 1989). In CPMV-infected cowpea protoplasts, the movement tubules occluding virions are extensively formed at the cell surface (van Lent et al., 1991), mimicking the process in plant tissue even in the absence of intact plasmodesmata. This material provides an opportunity for further identification and characterization of components involved in tubule formation. By analysis of deletion and insertion mutants and by transient expression experiments it was shown that the MP of CPMV was the sole viral protein responsible for tubule induction (Kasteel et al., 1993.The typical association between the CPMV movement tubules and the plasma membrane (in protoplasts and in plant tissue) lead to the speculation that one or more host components could be involved, either as a structural component of the movement tubule or in the process of anchoring of the tubule at the plasma membrane. As expression of the MP by a baculovirus expression vector resulted in identical tubule formation at the insect cell surface (Kasteel et al., 1996), it was Author for correspondence : Jan van Lent.Fax j31 317 484820. e-mail jan.vanlent!medew.viro.wau.nl suggested that host components, if involved at all in the tubule-forming mechanism, should be of a conserved nature (e.g. cytoskeleton proteins).To identify the major structural components of the movement tubule, tubular structures were purified from CPMV-infected cowpea protoplasts (Vigna unguiculata ' California Blackeye ') by means of differential centrifugation and analysed for their protein content by gel electrophoresis and immunoblotting. As tubule isolation from intact plant tissue is not feasible, infected protoplasts were used for mass production of tubules. In protoplasts, these tubules protrude from the cell surface and are easy to separate from other cell constituents.Protoplasts were isolated and inoculated with CPMV RNA or mock-inoculated with water as described by Eggen et al. (1989) and screened for infection and tubule formation by immunofluorescence microscopy using polyclonal antisera against CP and MP (Wellink et al., 1987),...