T. Godefroy-Colburn

Nucleic acid-binding properties of the alfalfa mosaic virus movement protein produced in yeast.

The movement protein of alfalfa mosaic virus (P3) was purified from yeasts transformed with an expression vector containing the P3 gene. Its nucleic acid-binding properties were tested by electrophoretic retardation, nitrocellulose retention, and RNA-protein cross-linking. The recombinant protein had a higher affinity for single-stranded RNA and DNA than for double-stranded nucleic acids. Each nucleic acid molecule bound several protein molecules without sequence specificity. The binding was 80% inhibited by 0.2 M NaCl. These properties are qualitatively similar, but not strictly identical, to those of two other viral movement proteins, the 30-kDa protein of tobacco mosaic virus and the gene I product of cauliflower mosaic virus.

A Non-structural Protein of Alfalfa Mosaic Virus in the Walls of Infected Tobacco Cells

Summary The 32000 mol. wt. (32K) non-structural protein of alfalfa mosaic virus, P3, has previously been detected in a crude membrane fraction of infected tobacco leaves, where it accumulated transiently at the beginning of the infection period. We show here, by immunoblotting with an antiserum to a synthetic peptide corresponding to the C terminus of the protein, that the majority of P3 is in the cell wall fraction where it remains throughout infection, both at 25 °C and at 10 °C. The cell wall-associated, P3-related material is heterogeneous and contains polypeptide species of slightly lower electrophoretic mobility than the major in vitro translation product of RNA 3, which suggests that P3 may be post-translationally modified.

In situ location of an alfalfa mosaic virus non-structural protein in plant cell walls: correlation with virus transport

Summary The 32000 mol. wt. non-structural protein (P3) of alfalfa mosaic virus (AlMV) has previously been shown to accumulate in the cell wall fraction of tobacco leaves infected with AlMV. We now report the ultrastructural location of this protein. P3 was visualized immunocytochemically in the middle lamella of the walls of those parenchymal or epidermal cells which had just been reached by the infection front and in which viral multiplication had just begun. P3 was not found when AlMV had accumulated to high levels in infected cells. These findings support the concept that P3 is involved in the spread of viral infection from cell to cell, i.e. is the transport factor of AlMV.

The Fate of the Transport Protein of Tobacco Mosaic Virus in Systemic and Hypersensitive Tobacco Hosts

Summary The transport protein of tobacco mosaic virus (TMV) (M r 30000, 30K non-structural protein) was detected on Western blots, using an antiserum to a synthetic C-terminal nonapeptide. Accumulation of this protein in subcellular fractions of inoculated leaves was measured during TMV infection of Nicotiana tabacum cv. Samsun and cv. Samsun NN. In cv. Samsun, a systemic host, the 30K protein appeared transiently in a crude membrane fraction but accumulated more stably in cell walls. In cv. Samsun NN, which is a hypersensitive host giving only localized infection, the early accumulation (up to 40 h; before any necrosis was visible) was the same as in cv. Samsun. However, as soon as necrosis was visible, the amount of 30K detected in the cell wall fraction decreased sharply and coat protein synthesis stopped. This drop in the amount of 30K protein is most easily interpreted as a side-effect of the hypersensitive reaction and may explain why TMV infection becomes localized in leaves of cv. Samsun NN.

Kinetics of accumulation of the three non-structural proteins of alfalfa mosaic virus in tobacco plants

Summary Antisera to synthetic peptides corresponding to the C-termini of two non-structural proteins (NSP) of alfalfa mosaic virus (AIMV), P1 (126K) and P3 (32K), were prepared and characterized. These antisera, together with one which had previously been made against the C-terminus of P2 (90K), enabled us to detect the three NSPs in a crude membrane fraction from AlMV-infected tobacco leaves. The accumulation of these proteins at 25 °C and at 10 °C was followed as a function of time after inoculation, and their amounts were compared with viral replicase activity. All three proteins accumulated at the beginning of the infection period and then disappeared, P3 more rapidly than the other two. There was a good correlation between replicase activity and the amounts of P1 and P2, but not the amount of P3. These results are consistent with the notion that P1 and P2 are part of the replication complex. Although much less coat protein was made in inoculated leaves at 10 °C than at 25 °C, the maximum amounts of the three NSPs and the maximum replicase activity were at least as high at 10 °C as at 25 °C. Thus, 10 °C is not a restrictive temperature for the assembly of a functional replication complex.

Effect of the alfalfa mosaic virus movement protein expressed in transgenic plants on the permeability of plasmodesmata

Symplastic transport of different sized fluorescent probes has been assessed in leaf epidermal cells of transgenic Nicotiana plants expressing the movement protein (MP) of alfalfa mosaic virus (AMV). In both N. tabacum and N. benthamiana, the size exclusion limit (SEL) of plasmodesmata increased from M(r) 1000, which represents the commonly accepted limit, to over 4.4K. However, in control plants, movement of a 3K probe was seen in 11 to 22% of the injections, indicating that plasmodesmata may on occasion allow the passage of molecules larger than was previously thought. The increase of SEL due to the presence of the AMV MP, although significant, remains insufficient to permit the passage of viral particles and the possibility of other mechanisms involved in viral cell-to-cell spread is discussed.

Mapping of the RNA-binding domain of the alfalfa mosaic virus movement protein

In-frame contiguous deletions were created in the movement protein gene of alfalfa mosaic virus by site-directed mutagenesis. The mutated movement proteins were expressed in Escherichia coli, extracted and then purified by denaturing gel electrophoresis and then renatured. Their binding ability with RNA was assayed by electrophoretic retardation and u.v.-crosslinking. Results indicated that a domain included within amino acids 36 to 81 was necessary for RNA binding.

An N-proximal sequence of the alfalfa mosaic virus movement protein is necessary for association with cell walls in transgenic plants

We have made transgenic tobacco plants (Nicotiana tabacum, cv. Xanthi nc) expressing the movement protein (P3, 300 amino acids) of alfalfa mosaic virus (A1MV) and two N-terminally deleted proteins lacking respectively 12 and 77 amino acids of the P3 sequence (P3 delta[1-12] and P3 delta[1-77]). The same proteins were expressed in recombinant yeast. By subcellular fractionation, the full-length P3 protein expressed by transgenic plants was found to be associated with cell walls as well as with cytoplasmic particulate material, as was the wild type movement protein expressed by A1MV-infected tobacco plants. P3 delta[1-12] behaved similarly but P3 delta[1-77] was found only in the cytoplasm. It thus appears that a polypeptide domain located between amino acids 13 and 77 of the P3 sequence is necessary for association of the protein with cell walls.

Binding of RNA by the alfalfa mosaic virus movement protein is biphasic

The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single‐stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated ‘primary complexes’) were detected by a nitrocellulose‐retention assay within 1 min of mixing, both At 4 and 22°C. In contrast, an incubation of 30 min at 22°C was necessary to obtain electrophoretically retarded complexes (‘stabilized complexes’), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4°C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a biomolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.

Preparation of an Antiserum against an in vitro Translation Product of Alfalfa Mosaic Virus RNA 3

Summary Antisera were raised against P3 (mol. wt. 32000), the full-length translation product of alfalfa mosaic virus (AlMV) RNA 3. P3 was made by translation in wheat germ extracts, using unfractionated AlMV RNA as message, and the products were then fractionated either by centrifugation through a sucrose gradient, or by phosphocellulose chromatography, each technique being followed by SDS-PAGE. Preparations made by each method were used in sequence to immunize a rabbit. The resulting antisera reacted on immunoblots with P3 synthesized in vitro, at dilutions of about 1:10000, but did not react with the translation products of AlMV RNA 1, RNA 2 or RNA 4. Although the antisera contained antibodies against some wheat germ components, most were removed by preabsorbing the antisera with the wheat germ extract. A minor wheat germ translation product of RNA 3, P′3, also reacted with the antisera. Since the mol. wt. of P′3 is apparently 1500 higher than that of P3, it is possible that P′3 is a readthrough product of the P3 cistron. With one antiserum preparation we were able to detect P3 in extracts of tobacco plants infected with AlMV.