No abstract

The expression and subcellular location of the 33.6-kDa nonstructural protein NSm of tomato spotted wilt virus (TSWV) was analyzed in Nicotiana rustica plants and protoplasts as a function of time. Immunofluorescent studies in protoplasts isolated from TSWV-infected N. rustica leaves showed that this protein could first be detected close to the periphery of the cell, near the plasmamembrane, and later in tubular structures emerging from the cell surface. In situ, these tubules appeared specifically in the plasmodesmata, suggesting their involvement in cell-to-cell movement of the virus during systemic infection. In protoplasts transfected with an expression vector containing the NSm gene, similar tubules were formed, indicating that NSm has the ability to form these structures in the absence of other virus-specific components. To test whether plant-specific components were involved in tubule formation, the NSm gene was also expressed in a heterologous expression system, i.e., insect cells. Spodoptera frugiperda and Trichoplusia ni cells were infected with a recombinant baculovirus expressing the NSm-gene (AcNPV/NSm). The efficient formation of NSm-containing tubules emerging from the surface of both cell types indicate that no plant-specific cell structures or proteins are involved in their development.

In cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the viruscontaining tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule 'grows' into a neighbouring cell in vivo. On average, 75 % of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 gm in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.

Electroporation has been shown to increase the potency of DNA vaccines that have demonstrated significant potential in mice. However, there is a need to develop noninvasive or minimally invasive vaccination methods. In pigs, in vivo gene expression was assessed to compare intradermal needle injection to a needle-free dermal BioJect as a means of delivery of plasmids. Each administration method was further tested with and without surface electroporation. Experiments with plasmid DNA encoding luciferase demonstrated that needle-free administration results in higher gene expression levels than needle injection. Electroporation enhanced gene expression for both intradermal delivery methods. Needle-free plasmid injection in combination with electroporation led to a more rapid induction of immune responses compared to other methods of plasmid administration. It was concluded that needle-free topical electroporation significantly enhances gene expression, possibly by improving cellular uptake of plasmid DNA.

A model for the maturation of tomato spotted wilt virus (TSWV) particles is proposed, mainly based on results with a protoplast infection system, in which the chronology of different maturation events could be determined. By using specific monoclonal and polyclonal antisera in immunofluorescence and electron microscopy, the site of TSWV particle morphogenesis was determined to be the Golgi system. The viral glycoproteins G1 and G2 accumulate in the Golgi prior to a process of wrapping, by which the viral nucleocapsids obtain a double membrane. In a later stage of the maturation, these doubly enveloped particles fuse to each other and to the endoplasmic reticulum to form singly enveloped particles clustered in membranes. Similarities and differences between the maturation of animal-infecting (bunya)viruses and plant-infecting tospoviruses are discussed.