Characterization of cowpea chlorotic mottle virus and its assembly

This thesis decribes the conditions for isolation of cowpea chlorotic mottle virus (CCMV), its ribonucleic acid (RNA) and the coat protein, the characterization of the virus and its constituents (chapter 3, 4 and 5) and the dissociation and assembly behaviour of the virus (chapter 6 and 7).

The aim of the investigation and a literature review pertaining to RNAprotein interactions, which are met with the tobamoviruses, the Leviviridae and the bromoviruses are given in chapter 1 and 2.

CCMV, isolated and purified in the prescence of reducing agents such as ascorbic acid and mercaptoethanol, contained variable amounts of degraded RNA. At first RNA-2 was cleaved into two fragments but subsequently all RNA molecules were cleaved at random sites. The buoyant density in RbCl and the sedimentation coefficient of the virus remained unchanged i.e. the degraded RNA was still bound to the protein coat and did not change the stability of the nucleoprotein particles.

The degradation of the RNA was stimulated when virus was incubated at 37°C in the presence of reducing agents such as mereaptoethanol. Besides reducing agents also oxygen and traces of metals appeared to play a role in the degradation process. Addition of chelating agents, such as 1 mM EDTA, to the homogenization buffer and the buffers in which the virus was kept, prevented in situ RNA degradation. Problably the degradation is caused by radicals, which are formed during the auto-oxidation of reducing agents by oxygen, catalysed by traces of metals (chapter 3).

In chapter 4 a describtion is given of three coat protein isolation methods and the influence of the isolation method on the formation of pseudo top component (PT) i.e. an empty protein shell without RNA. By means of CaCl 2 RNA free coat protein could be isolated from virus, even when the virus particles contained exstensively degraded RNA. The formation of PT and its dissociation were pH dependent and both processes showed a remarkable hysteris effect. This effect can be explained by assuming two stable conformations of the coat protein.

In chapter 5 the results of partial specific volume, ciruclar dichroism (CD) and sedimentation equilibrium measurements of CCMV are given. The apparent partial specific volume of the dissociated protein in 0.5 M CaCl 2 pH 7.5, mainly the dimer of the coat protein subunit, changed from 0.737 cm 3/g to 0.728 cm 3/g in 0.2 M NaCI, 0.01 M CaCl 2 pH 5.0, mainly PT. Calculation of the partial specific volume of CCMV from the experimentally determined volumes of RNA, 0.476 cm 3/g and coat protein, 0.745 cm 3/g in 0.2 M NaCl. 1 mM EDTA pH 5.0 resulted in a value of 0.660 cm 3/g, which is lower than the experimentally determined partial specific volume of CCMV, 0.719 cm 3/g. The difference is caused by RNA-protein interaction.

The CD measurements of protein dimers and PT showed little difference between the secondary structure of both protein subunit aggregates. The alpha-helix content was in both cases smaller than 1%. The structure of the RNA, both free in solution and inside the virus particle showed a large amount of base pairing and base stacking. Small changes in the secondary structure occured. when the virus was swollen and dissociated.

Chapter 6 describes the pH and ionic strength dependent dissociation of CCMV. Upon increasing the pH from 5.0 to 7.5 at 1 M NaCl, CCMV formed RNA-protein complexes, which sedimented slower than the intact virus particles but still retained an RNA-protein ratio identical to virus. When CCMV was incubated at pH 7.5 with increasing concentrations of NaCl, at first unfolding of the RNA occured, while all the protein subunits were still bound to the RNA, followed by a gradual release of protein subunits. In 1 M NaCI the RNA retained 4 to 8 protein subunits per RNA molecule. This RNA-protein complex is probably involved in the recognition of the protein by the viral RNA.

In chapter 7 is described how this RNA-protein complex has been used for assembly of virus particles, 90% of which is stable in RbCl. These particles obtained after dissociation and reassociation of CCMV were characterised with respect to RNA content and compared with virus particles obtained after assembly of isolated RNA and coat protein.

After centrifugation in a sucrose gradient both reassociated and assembled virus showed a band with a sedimentation coefficient of about 80 S at the main product. These particles contained RNA-1 and -2, comparable to the original virus preparation but less RNA-3 and -4. Two other classes of products were observed. On one hand a fraction sedimenting between 70 and 80 S, which contained mainly particles with RNA-3 and on the other hand a fraction with a sedimentation coefficient>110 S, in which some particles with RNA-1 and -2 occured, but mainly particles with RNA-3 and -4. Only 40% of the nucleoprotein particles assembled from isolated RNA and protein appeared to be stable in RbCl.

Probably RNA-1 and RNA-2 can form stable virus particles by means of the RNA-protein complex, while RNA-3 also has to make a link with RNA-4, before a stable nucleoprotein particle sedimenting at 80 S is formed. The assembly products of CM are compared in this chapter with those of broad bean mottle virus and brome mosaic virus, two other bromoviruses.

Possible assembly mechanisms, a model for the coat protein dimer and future assembly research are discussed in chapter 8.