Protein synthesis directed by cowpea mosaic virus RNAs

The study described here concernes the proteins that are synthesized under direction of CPMV-RNAs.In chapter III it is described that sufficient radioactive labelling of proteins was achieved when 35 S as sulphate was administered to intact Vigna plants, cultivated in Hoagland solution. After fractionation of the cells, a fraction containing virus-specific (vesicle) structures could be isolated in which the majority of the replicase activity and CPMV-specific dsRNA was located. Comparison of proteins extracted from these fractions from infected and mock-infected leaves resulted in the detection of twoproteins, besides the coat proteins. These proteins, with molecular weights of about 170,000 and 72,000 could only be detected after the development of the radioactive labelling technique using 35 S as sulphate.In vitro studies using the messenger-dependent lysate from rabbit reticulocytes (MDL) and wheat germ extracts show that the virion RNAs stimulate the incorporation of amino acids in acid precipitable material. The optimization and characteristics of both in vitro systems are described in chapter IV.Product analysis of the synthesized polypeptides shows that the CPMV-RNAs can be reproducibly translated into well defined products (chapter V), indicating that these RNAs act well as messengers in the cell-free systems used. From the characteristics of these systems and the product analysis, it has become clear that CPMV-RNAs needed particular conditions for their in vitro translation (chapter IV and V). In wheat germ extracts the addition of spermidine (0.4 mM) together with a lowering of the Mg 2+ concentration to about 3.0 mM was required for the efficient translation of CPMV-RNAs. Furthermore, increasing the K + concentration to 130 mM was advantageous for the translation into high molecular weight polypeptides. In the MDL the addition of heterologous tRNA was absolutely necessary for the translation of CPMV-RNA into high molecular weight products.Estimations of the molecular weights by polyacrylamide gel electrophoresis in SDS containing gels, of the products synthesized, showed that there is a considerable variation in the molecular weights determined. The molecular weight of BP (the largest product synthesized under direction of B-RNA) was estimated to be 180,000 to 190,000. MP 1 (the large molecular weight product synthesized under direction of M RNA) has a molecular weight that varies from 110,000 to 125,000. The other largepolypeptide synthesized under direction of M-RNA (MP 2 ) has an estimated molecular weight between 105,000 and 95,000.Comparison between the polypeptides synthesized in both types of extract shows that the products are - at least electrophoretically - identical (chapter VI). Also in chapter VI it is described that comparison with virus-specific proteins found in vivo gives little information about either the origin of the virus-specific proteins found in vivo or about the nature of the in vitro synthesized polypeptides.In all the in vitro translation experiments no coat proteins could be detected among the translation products. In chapter VII experiments are described which were undertaken to reveal the translation strategy of CPMV-RNAs. From these experiments it was concluded that it was very unlikely that the coat proteins are synthesized from subgenomic messengers nor from internal initiation sites. The overall conclusion is that the large polypeptides synthesized under direction of Band M-RNA are probably precursor molecules from which the coat proteins (and other virus-specific proteins) are generated by a mechanism of posttranslational cleavage. Processing experiments in either wheat germ extracts or MDL did not, up until now, reveal any precursor- product relations hip between the polypeptides synthesized. This is in contrast to other findings where in MDL the M-RNA specific products could be processed (Pelham, Virology 96, 463), albeit into products not recognized as being virus-specific proteins.