Characterization of a defective form of tomato spotted wilt virus

The work described in this thesis was aimed at the elucidation of the nature of a defective form of TSWV which does not form complete particles during infection.Properties of TSWV and the existence of a defective form of this virus are described in Chapter 1. A survey of the literature on three different types of defective viruses with properties significant for the understanding of the defect in TSWV is given also in this chapter. The purposes of the study are presented at the end of this chapter.In view of a defect in the genome of TSWV experiments were performed to determine the exact number of RNA segments in normal TSWV particles and the coding function of each individual RNA segment (Chapter 2). Electrophoresis of TSWV RNA on denaturing agarose gels revealed three segments with mol. wts. of 2.7, 1.7 and 1.1 x 10 6, to which will be referred as RNA 1, 2 and 3.TSWV RNA acted as messenger in a cell-free system of wheat germ and in a mRNA-dependent rabbit reticulocyte lysate, indicating that it is of a positive strand. Analysis of the in vitro translation products by gel electrophoresis revealed that two major polypeptides were synthesized in both systems. One could be indentified by immunoprecipitation as the nucleocapsid protein; the identity of the other polypeptide, having a mol. wt. of 60 000, is unknown.The positive strandness of TSWV RNA could be confirmed by other experiments. TSWV RNA labelled with 125I did not hybridize with polyribosomal RNA from infected leaves and transcriptase activity could not be detected in purified preparations of TSWV or in nucleocapsid extracts from infected leaves.In vitro translation of the RNA of TSWV fractionated on sucrose gradients, and analysis of the translation products revealed that the nucleocapsid protein and a protein with a mol. wt. of 60 000 is encoded for by RNA 3. The coding functions of the two other RNA segments could not be determined.The viral proteins present in plants infected with defective isolates were analysed by serological means (Chapter 3). Antibodies were raised against the nucleocapsid protein and against a fraction containing the membrane proteins of TSWV Sap from leaves infected with isolates either of the normal or the defective form were tested in ELISA. Antibodies directed against the nucleocapsid protein reacted with sap from leaves infected with isolates of both forms. Antibodies directed against the membrane proteins reacted only with sap from leaves infected with normal isolates and not at all with sap from leaves infected with defective isolates. These results indicate that the defective form does not direct the synthesis of membrane proteins and consists solely of nucleocapsids.Chapter 4 and 5 describe the analysis of the RNAs which are synthesized in leaves upon infection with either the normal or defective form of TSWV Polyribosomes were isolated from infected plants at different times of infection. They were allowed to complete nascent polypeptide chains in an in vitro translation system and the synthesized polypeptides were analysed by polyacrylamide gel electrophoresis. Synthesis of the nucleocapsid protein in the course of infection with isolates of both forms could be detected. However, synthesis of the other viral structural proteins or proteins other than the polypeptides synthesized under direction of polyribosomes from healthy plants, could not be observed (Chapter 4).The RNAs synthesized upon infection were also analysed by hybridization (Chapter 5). The three individual TSWV RNA segments were labelled with 32P and hybridized with dsRNA isolated from plants infected with isolates either of the normal or defective form. RNA 1 and RNA 3 hybridized to the same extent with dsRNA from plants infected with isolates of both forms. RNA 2 hybridized to a significant lower value with dsRNA from plants infected with the defective isolate. This result indicates that in plants infected with the defective isolate the genetic information of RNA 1 and RNA 3 of normal TSWV particles is present, but the information of RNA 2 is only partly present. Since the defective form does not direct the synthesis of membrane proteins, RNA 2 contains possibly the information for the membrane proteins.Chapter 6 describes experiments on the nature of the electron dense amorphous masses, by which the defective form is defined electron microscopically. An infectious nucleoprotein-rich fraction was purified from plants infected with several defective isolates. Analysis by ELISA and polyacrylamide gel electrophoresis revealed the presence of nucleocapsid protein of TSWV in this fraction. NO other viral structural proteins were found. Electron microscopic examination of this fraction demonstrated structures resembling the electron dense amorphous masses. As this fraction was infectious it is concluded that the characteristic amorphous masses are composed of nucleocapsids. Analysis by gel electrophoresis of RNA extracted from the fraction revealed that the three defective isolates of TSWV studied have an RNA 1 and RNA 3 with the same mobility as those of normal TSWV particles. RNA 2 was much smaller, indicating that RNA 2 of the normal form has lost a fragment. The same conclusion was also obtained in experiments in which the viral RNA segments were hybridized with dsRNA (Chapter 5).The results of this study are discussed in Chapter 7. The nature of the defect in TSWV giving rise to the defective form is defined and the significance of the results in view of the coding functions of the three RNA segments of TSWV is discussed. Also, the defective form of TSWV is compared with the defective forms of other viruses and the possible role of the viral envelope in the transmission of TSWV by thrips is discussed.