L. Hirth

Evidence for polar reconstitution of TMV.

During reconstitution experiments conducted with Tobacco Mosaic Virus (TMV), Stussi, Lebeurier and Hirth [ 1 ] described incompletely coated TMV particles (IC) of 700 A length. It was demonstrated that the free RNA of these particles was able, under certain conditions, to be coated and to give infectious TMV particles. These observations suggested a polar process of reconstitution. But conflicting results were reported on this subject by several research workers. Fraenkel-Conrat [2] was unable to demonstrate by chemical means the polarity of selfassembly in the case of TMV. On the other hand, Butler and Klug [3] and Guilley, Stussi and Hirth [4] by controlled digestion of TMV-RNA with exonucleases, indicated that reconstitution of TMV began by the SOH end. However, difficulties exist in the use of exonuclease and some ambiguities appeared in the interpretation of the results. The present paper reports experiments which suggest strongly that TMV protein recognises the 5’OH end of the TMV-RNA and that reconstitution is a polar process.

Translation of tobacco rattle virus RNA in vitro using wheat germ extracts

Abstract When added to extracts from commercial wheat germ, tobacco rattle virus (TRV) RNA stimulated incorporation of radioactive amino acids into protein with an efficiency approaching that of tobacco mosaic virus (TMV) RNA. RNA from the smaller particle (RNA-2) of the CAM strain of TRV was translated largely into a single polypeptide which coelectrophoresed with coat protein and aggregated specifically with unlabeled protein. Coelectrophoresis with coat protein in 3% acrylamide gels indicated a C-terminal sequence in the radioactive product similar to that in coat protein. Attempts to change the pattern of translation products by heating RNA, adding coat protein to incubations, or changing RNA concentration were unsuccessful. RNA from the larger particle (RNA-1) of strain CAM (Campinas) was translated into a variety of products with a maximum molecular weight of 100,000. When mixtures of RNA-1 and RNA-2 were used, RNA-2 was translated preferentially.

Further studies on the translation products of tobacco rattle virus RNA in vitro.

Abstract Translation of RNA-1 (MW, 2.5 × 106) of tobacco rattle virus (TRV) strain PRN in wheat germ extracts containing spermidine resulted in the production of many polypeptides with a maximum MW of 170,000 and a clear band of 140,000. Translation of RNA-1 in lysates of rabbit reticulocytes resulted in these two products but little else. PRN-RNA-2 (MW, 1.0 x 106) was translated in both in vitro systems as two polypeptides, coat protein and a protein of MW 31,000 (band 1). Three bands of possible premature termination products were also produced in wheat germ extracts. The identity of coat protein was shown by coelectrophoresis in 10 and 3% polyacrylamide gels, specific aggregation with PRN protein, and coincidence between tryptic peptide fingerprints. Tryptic peptide mapping showed that band 1 was not a precursor for coat protein. There were, however, some peptides in digests of band 1 which coincided with coat protein peptides, which suggests a partial overlap of amino acid sequences. The relative abundance of coat protein and band 1 protein in translation products of PRN-RNA-2 in wheat germ extracts was dependent on the concentration of magnesium ion in the extracts. At concentrations suboptimal for total incorporation, translation resulted mainly in coat protein, whereas, at supraoptimal concentrations of magnesium, band 1 protein predominated in the translation products. We suggest that PRN-RNA-2 contains two open initiation sites.

Phenylalanine ammonia-lyase in tobacco mosaic virus-infected hypersensitive tobacco. Density-labelling evidence of de novo synthesis.

A strong increase in phenylalanine ammonia-lyase (EC 4.3.1.5) activity occurs in tobacco mosaic virus-infected tobacco leaves developing necrotic local lesions. Comparison of physicochemical properties of the partially purified enzymes extracted from healthy and infected leaves showed that the hypersensitive reaction leads to an increase in the pool size of the same active enzyme molecules as those present in non-infected material. The molecular mechanism of enzyme formation was investigated by radiolabelling with [3H]leucine and by density labelling with 2H2O. Abnormal patterns of incorporation of radioactivity into all soluble proteins were found in infected leaves carrying local lesions. In contrast, uptake of deuterium into the amino acid pool was the same in healthy and infected leaves. Unstimulated phenylalanine ammonia-lyase was shown to be a long-lived enzyme (half-life: 25-35 h). Results of comparative density labelling experiments unequivocally demonstrated that the increased enzyme pool size arose from an increased rate of synthesis mediated by the hypersensitive reaction.

Specificity of in vitro reconstitution of bromegrass mosaic virus

Bromegrass mosaic virus (BMV) RNA was allowed to compete with yeast tRNA or alfalfa mosaic virus (AMV) RNA for in vitro encapsidation by BMV protein. Various proportions of 32P-labeled foreign RNAs were added to a reassembly mixture (BMV protein: BMV RNA, 4:1) and the reassembly products were observed by analytical and rate-zonal sedimentation, and the RNA contents of the nucleocapsids were examined by gel electrophoresis. Incubation of BMV protein with tRNA alone produced 56 S particles containing five or six tRNA molecules per particle, but with both tRNA and BMV RNA present very little of the tRNA was incorporated. AMV 12 S RNA led to 64 S particles containing one AMV RNA molecule: with both AMV and BMV RNAs present, the smallest BMV RNA outcompeted the AMV RNA about fourfold, even though the two RNAs have similar molecular weights and biological functions. Evidently BMV protein does to some extent specifically recognise its own RNA molecules.

The uncoating of native and reconstituted TMV by dimethylsulphoxide: the polarity of stripping.

The effect of increasing concentrations of dimethylsulphoxide (DMSO) on the stability of native and reconstituted tobacco mosaic virus (TMV) has been investigated by means of electron microscopy and infectivity assay. The removal of protein subunits from TMV by DMSO was found to be a stepwise process. Subunits are first removed from the 3OH end of the rod but at about 72% DMSO the site at which uncoating occurs shifts to the 5 end. Studies with reconstituted and partially reconstituted TMV confirm the stepwise nature of the uncoating process. Complete uncoating of reconstituted TMV was found to occur at smaller DMSO concentrations than that of native TMV.

Fluorescence study of tobacco mosaic virus protein

Abstract Protein fluorescence properties of tobacco mosaic virus [3 Trp residues per monomer (positions 17, 52, 152)] and of two tobacco mosaic virus mutants [green tomato atypical mosaic virus, 2 Trp (52, 152) and cucumber virus 4 , 1 Trp (unknown position)] have been studied. Emission spectra, fluorescence quantum yields and lifetimes were determined. Results showed that protein fluorescence is due to buried Trp only, except for the cucumber virus 4 strain, in which Tyr also contributes to the emission. Comparison of the three strains showed that Trp 17 and Trp 52 have high fluorescence yields ( ϕ 17 = 0.29; ϕ 52 = 0.37) whereas Trp 152 (probably present in cucumber virus 4 ) is strongly quenched ( ϕ 152 = 0.035). An unusually efficient Ayr → Trp energy transfer was observed in tobacco mosaic virus protein, indicating that most of four Tyr residues are located near the highly fluorescent Trp.

Specificity of TMV RNA Encapsidation: in Vitro Coating of ,Heterologous RNA by TMV Protein

Abstract Tobacco mosaic virus (TMV) RNA and TMV coat protein were reconstituted in the presence of an excess of host RNA or turnip yellow mosaic virus (TYMV) RNA. TMV protein and its homologous RNA recognized each other specifically in the presence of an excess of host RNA, but when TMV RNA and TYMV RNA were mixed with protein, both RNAs were encapsidated. Indeed, at pH 6, TYMV RNA was recognized by TMV protein at least as well as TMV RNA itself. This suggests that TYMV RNA like TMV RNA may possess a special site recognized by TMV protein.

In Vitro encapsidation of the four RNA species of brome mosaic virus

Abstract The products of in vitro reconstitution of brome mosaic virus (BMV) protein and RNA were studied using analytical sedimentation and polyacrylamide gel electrophoresis. The product formed depended on the protein concentration, even though the ratio of RNA to protein was constant (1:4). At a low ionic strength with protein at 0.2 g/liter, mostly 78 S particles (like native BMV) were made, whereas at 2 g/liter of protein, reconstitution proceeded erratically. When the ionic strength was decreased to various levels by dialysis after mixing RNA and protein at I = 1.0, the four RNAs were encapsidated at different rates according to species, with RNAs 4 and 3 at the highest rates. The same sequential encapsidation in order of increasing molecular weight occurred as the ratio of protein to RNA was increased. Some particles containing three copies of the smallest RNA were found. These observations show that BMV protein displays in vitro a differential affinity for the various BMV RNA species.

Rapid labelling of a nonencapsulated RNA of bromegrass mosaic virus.

Recently Lane and Kaesberg [l] have observed that bromegrass mosaic virus (BMV) contains 5 types of RNA. The mixture of the three largest RNA’s is infectious while the smallest RNA and a minor component seem to have no effect on infectivity. We report in this paper on a study of the incorporation of j2P into the different RNA’s of BMV. We have found that the different RNA’s are labelled and encapsulated at different rates.