Viral protein synthesis in cowpea mosaic virus infected protoplasts

P. Rottier

Research output: Thesisinternal PhD, WU


<p/>In contrast to the situation concerning bacterial and, to a lesser extent, animal RNA viruses, little is known about the biochemical processes occurring in plant cells due to plant RNA virus infection. Such processes are difficult to study using intact plants or leaves. Great effort has therefore been spent in developing <em>in vitro</em> cultures of plant protoplasts, but the use of these protoplasts has been seriously hampered by various technical problems.<br/>It is clear that plant RNA virus infections give rise to a number of specific reactions in infected cells, particularly at the level of RNA and protein synthesis. The multiplication of these viruses implies a manifold copying of their structural components, namely RNA and protein. These activities clearly require the involvement of other RNA and protein species in view of, for instance, the drastic cytopathic effects that are often caused by infection. Thus, detailed knowledge of virus-induced RNA and protein synthesis is a prerequisite for an understanding of the infection process.<br/>In this thesis investigations are described concerning the proteins involved in the multiplication of cowpea mosaic virus (CPMV) in cowpea protoplasts.<br/>The relevant properties of CPMV are summarized in chapter 2. Research on CPMV is interesting not only in the field of plant virology as the virus is also of general virological significance. Structurally, for example, CPMV shares a number of striking features with the animal picornaviruses like poliovirus and footand-mouth disease virus. From a molecular biological point of view the multiplication of CPMV presents several intriguing specific questions. CPMV is a multicomponent virus: its genome consists of two different single-stranded RNA molecules occurring in separate nucleoprotein particles, both of which are essential for infection. It would be of interest to learn which functions are specified by the two genome pieces, whether the separate viral components can give rise to some virus-specific biochemical expression within the cell and how they complement each other during normal infection. Characteristic cytopathic structures are induced in plant cells upon infection with CPMV in the form of vesicular membranes embedded in amorphous electron-dense material. It is now well established that replication of the viral RNA is localized in these structures. Information about their development and functioning is thus required.<br/>Chapter 2 also contains an introduction to the essential features of plant protoplasts and an enumeration of the diverse possibilities they can offer for the investigation of specific virological problems. Finally, the present state of knowledge about the biochemistry of multiplication of plant viruses, as obtained mainly through the use of protoplasts, is reviewed.<br/>The isolation of viable protoplasts from the leaf mesophyll tissue of cowpea requires the stringent control of the growth conditions of the plants (chapter 3). Various factors appeared to influence the quality of the protoplasts and their infectability with CPMV. The combination of cowpea mesophyll protoplasts and CPMV appeared to offer a very attractive system. Based on the original study of HIBI <em>et al.</em> (1975) <em></em> a procedure was developed for the preparation and infection of the protoplasts, the most rapid and simple one so far described (chapter 3). The major reasons for this are:<br/>- a short growth period (about 10 days under the conditions used) is required to obtain suitable leaf material<br/>- for an efficient enzymatic digestion of the leaves it is sufficient to damage the lower epidermis by means of carborundum powder and paint-brush instead of stripping off the epidermis with forceps<br/>- enzymatic one-step isolation of protoplasts can be achieved by simultaneous digestion with pectinase and cellulase<br/>- inoculation of cowpea protoplasts with CPMV does not require the use of polycations, making the preincubations of protoplasts and virus superfluous.<br/>Using this standard procedure, synchronous infection in virtually all protoplasts could be routinely established.<br/>Radioactive precursors of RNA and protein added to the cowpea protoplast incubation medium were taken up and incorporated into macromolecules to high specific activities (chapter 4). This light-dependent metabolic activity increased during the first 30 h after isolation of the protoplasts. It could be influenced by several metabolic inhibitors. RNA synthesis, for instance, was strongly inhibited by actinomycin D and cordycepin, whereas ot-amanitin and rifampicin had only a slight effect. Protein synthesis in the protoplasts could be blocked completely by means of cycloheximide, and was also sensitive to the action of antibiotics such as chloramphenicol, puromycin and, to a lesser degree, lincomycin. These inhibitor studies, as well as the results of analyses of RNAs and proteins synthesized in the protoplasts, indicated that the metabolic activity of the chloroplasts is low and mainly limited to light-dependent energy generation.<br/>The multiplication of CPMV in cowpea mesophyll protoplasts relies upon host-coded DNA-dependent RNA synthesis during the earliest stage of infection. As described in chapter 5, <em></em> propagation of the virus was completely prevented when protoplast DNA transcription and thereby almost all RNA synthesis was inhibited by means of actinomycin D, provided that the drug was applied at the time of inoculation or immediately thereafter. The degree of inhibition of CPMV replication by actinomycin D rapidly decreased when the antibiotic was added later after inoculation; the production of viral nucleoprotein particles became progressively more resistant to the drug, showing complete resistance at about 8 h after inoculation, a time at which infection was still in its latent phase.<br/>Surprisingly, synthesis of CPMV antigen was still demonstrable by immunofluorescent antibody staining techniques in protoplasts in which virus replication had been completely blocked by actinomycin D. Under these circumstances <em>de novo</em> production of viral top component (empty capsids) was observed. In addition to the CPMV capsid proteins, these experiments revealed another 3 viral-related polypeptides in infected protoplasts (molecular weights 170,000, 110,000 and 84,000). Since their synthesis also clearly continued during inhibition of host RNA synthesis by actinomycin D it was concluded that they were coded for by the viral genome.<br/>A more detailed analysis of the proteins involved in CPMV multiplication is described in chapter 6. At least 11 proteins not present in mock-infected protoplasts or present in much lower amounts were detected in infected protoplasts. Their molecular weights were estimated to be 170,000, 130,000, 112,000, 110,000, 87,000, 84,000, 68,000,37,000, 30,000,24,000 and 23,000. Host-specific protein synthesis appeared to be hardly affected by C~ infection. Virusspecific proteins therefore had to be distinguished from a large variety of protoplast proteins. This appeared to be greatly facilitated by radioactive labeling of the protoplasts for short periods during a suitable phase of the infection cycle and by subcellular fractionation of the protoplasts. The treatment of the particulate fractions with the mild detergent digitonin yielded extracts in which proteins could be detected by polyacrylamide gel electrophoretic analysis that otherwise remained hidden by a general high background of radioactivity upon direct analysis of the untreated particulate fractions.<br/>All of the proteins apparently concerned in CPMV infection were most clearly observed late in infection. None of them was detectable during the actinomycin D-sensitive latent period. The 170 and 30 kilodalton proteins were the first detectable, namely from about 10 h after inoculation. All others became apparent about 6 h later.<br/>The induction of the characteristic cytopathic structures in CPMV-infected cells suggests the induction and/or stimulation of synthesis of a large number of proteins by the virus. Upon fractionation these proteins should appear in the particulate fractions of the protoplasts. However, only 2 virus-induced polypeptides (112 and 68 kilodalton) were demonstrated exclusively in these fractions. Other components of the cytopathic structures presumably remained in the extracted pellet residues.<br/>In chapter 8 further characterization of the viral-related proteins found in CPMV-infected protoplasts is described. The 37, 24 and 23 kilodalton proteins were identified as viral capsid proteins by comparing their electrophoretic mobility in polyacrylamide gels with those of proteins from purified CPMV and by immunoprecipitation with anti-CPMV serum. They occurred in the soluble fraction of protoplasts only in viral particles, not as free proteins. The 170 and 30 kilodalton polypeptides also appeared to be virus-specific and to be coded for by CPMV bottom component RNA: they comigrated in polyacrylamide slabgels with the main products of <em>in vitro</em> translation of this RNA in a messengerdependent lysate of rabbit reticulocytes. These polypeptides were also induced in protoplasts after inoculation with purified CPMV bottom component. Under these conditions some other viral-related proteins were also synthesized, but not the viral capsid proteins. In contrast, inoculation with purified CPMV middle component did not delectably affect protoplast protein synthesis. These experiments strongly suggest that CPMV B-RNA specifies one or more early functions one of which being the replicase function, while M-RNA specifies late functions, particularly the viral capsid proteins.<br/>Since the total size of the viral-related proteins by far exceeded the theoretical coding potential of the CPMV genome, the occurrence of precursor-product relations was investigated by means of pulse-chase experiments and by attempting to accumulate possible precursors by blocking their proteolytic processing. The results of the latter method were negative. Upon chasing the <sup>35</sup> S-methionine label with unlabeled amino acid the 87,000 dalton polypeptide appeared to be unstable, as was a protein with the same electrophoretic mobility synthesized in uninfected protoplasts. This protein thus probably represents a host-specific protein the synthesis of which is stimulated by CPMV infection. Furthermore, these experiments revealed that several discrete oligopeptides were cleaved from the smaller CPMV coat protein after assembly into a virion. Although the involvement of precursor- product relations could not be definitely excluded on the basis of the methods used, another possible mechanism of viral gene expression was also investigated, namely the occurrence of subgenomic viral messengers. Upon analysis of <sup>32</sup> P-labeled RNA from CPMV-infected protoplasts by polyacrylamide gel electrophoresis, 2 RNAs of genome length were the only detectable virus-specific polynucleotides. These results suggest a mechanism for the translation of the CPMV genome using internal initiation sites. Further research is, however, required to unequivocally elucidate the strategy of the viral gene expression.<p/>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • van Kammen, A., Promotor, External person
Award date21 Mar 1980
Place of PublicationWageningen
Publication statusPublished - 1980


  • cowpea mosaic virus
  • viruses
  • replication
  • plant diseases
  • plant pathology
  • malformations
  • fasciation
  • plant composition
  • protein synthesis

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