Aggregation of TMV CP plays a role in CP functions and in coat-protein-mediated resistance

Abstract

Tobacco mosaic virus (TMV) coat protein (CP) in absence of RNA self-assembles into several different structures depending on pH and ionic strength. Transgenic plants that produce self-assembling CP are resistant to TMV infection, a phenomenon referred to as coat-protein-mediated resistance (CP-MR). The mutant CP Thr42Trp (CP(T42W)) produces enhanced CP-MR compared to wild-type CP. To establish the relationship between the formation of 20S CP aggregates and CP-MR, virus-like particles (VLPs) produced by TMV variants that yield high levels of CP-MR were characterized. We demonstrate that non-helical structures are found in VLPs formed in vivo by CP(T42W) but not by wild-type CP and suggest that the mutation shifts the intracellular equilibrium of aggregates from low to higher proportions of non-helical 20S aggregates. A similar shift in equilibrium of aggregates was observed with CP(D77R), another mutant that confers high level of CP-MR. The mutant CP(D50R) confers a level of CP-MR similar to wild-type CP and aggregates in a manner similar to wild-type CP. We conclude that increased CP-MR is correlated with a shift in intracellular equilibrium of CP aggregates, including aggregates that interfere with virus replication.

Figure 1

Structural models of helical (a–c) and non-helical (d–f) assemblies of TMV CP^T42W. Panels a and d represent the molecular surfaces of two disks of helical and non-helical assembly of the protein, respectively. Images are colored according to electrostatic potential with positive and negative charges represented by blue and red, respectively. Panels b and e, show the general locations of the T42W mutation (highlighted by the white circle) in the helical and non-helical assemblies, respectively. Note that there is significantly more open space between the non-helical stacked disks than in the helical fibers. Each subunit of the aggregate is represented by a differently colored C-α backbone. Panels c and f are stereo images showing the environments of a.a. 42W in the helical and non-helical aggregates, respectively. The residues in contact with 42W are colored according to atom type with carbon, nitrogen, and oxygen colored yellow, blue, and red, respectively. The side chains for the 42W residue are represented by white stick models. Note that the 42W side chain environment is more crowded and hydrophilic than observed in the stacked disk conformation.

Figure 2

Histogram of the distribution of sizes of the particles. Comparison of the distribution of sizes of particles of TMV-CP^T42W between of particles and immuno-captured particles.

Figure 3

Cryoelectron microscopy of purified particles. a, c, c′ and e virus-like particles of TMV-CP^T42W; b, d and f virions of TMV. a, 10000X magnification; b and c, 25000X magnification. c′, Shows an enlargement of c, the arrow points to the stacked-disk like structure in the ’angled joints’ described in the text. d and e, Fast Fourier Transformation images. The black squares on panels b and c indicate the areas used in the FFT.

Figure 4

Inmuno-gold labeling of purified particles using Mab #16. a) TMV particles: left panel shows a particle with a single labeled end; the panel on the right shows label within the particle, see arrows. b) TMV-CP^T42W particles: left panel shows a particle with a single end-label, and the panel on the right shows labeling within the particle. c) TMV-CP^T42W particles with both ends labeled. The bar represents 100 nm.

Figure 5

Sucrose gradient analysis. Leaf homogenates were prepared from infected plants and analyzed on linear 5 to 40% sucrose gradients. Gradients were collected in 8 fractions plus pellet, and aliquots were subjected to PAGE in SDA and western blot analysis using anti-TMV serum. T= top of the gradient, B= bottom and P = pellet.