Cloned DNA species complementary to the RNA of tomato bushy stunt virus (TBSV) were produced and the location of each on the virus genome was determined. Polysomes were prepared from TBSV-infected plants and RNA, released from the polysomes by treatment with puromycin, was shown by Northern hybridization to contain TBSV genomic RNA (4.8 kb) and two subgenomic ssRNA species of 2.0 and 0.9 kb. Hybrid selection using clones corresponding to different parts of the virus genome showed that the two subgenomic RNA species are probably 3′ coterminal with the genomic RNA. In vitro translation of hybrid-selected RNA showed that the genomic (4.8 kb), 2.0 and 0.9 kb RNA species encode proteins of M r 37000 (37K), 40K (the virus coat protein) and 22K respectively. Translation in the presence of calf liver tRNA produced, in addition to the other products, a protein of M r 90K which may be a readthrough product of the 37K protein. The results have enabled a model for the genome organization and expression of TBSV to be formulated.
Recovery of highly purified citrus cachexia viroid (CCaV) was accomplished by serial elution following CF-11 cellulose chromatography of a 2 m-LiCl-soluble nucleic acid preparation. The alternative herbaceous host, cucumber (Cucumis sativus cv. Suyo), yielded greater quantities of the viroid than the highest yielding citrus host, citron (Citrus medica cv. Etrog). A randomly primed cDNA probe to CCaV purified from cucumber reacted positively to extracts from citron and cucumber inoculated with the same isolate of CCaV. When tested against a broad range of other citrus viroids, the CCaV cDNA hybridized to only one, CV-IIa, which has been identified as the causal agent of a mild form of the citrus exocortis disease. Because of the apparent homology between the nucleotide sequences of CV-IIa and CCaV, and a size difference of only five to ten nucleotides, these RNAs can be considered as members of a common subgroup of citrus viroids. These two viroids have been classified by bioassay reactions as the causal agents of two distinct types of citrus disease, an ‘exocortis-like’ syndrome and cachexia. The properties of and relationships between these two members of the citrus viroid II group and the definition of the exocortis and cachexia (xyloporosis) diseases are presented.
Nucleic acid extracts from citrons (Citrus medica cv. Etrog) displaying mild and moderate symptoms associated with the exocortis disease were analysed by sequential and denaturing PAGE which revealed the presence of several viroids. A comparison was made of electrophoretic patterns displaying one or more distinct citrus viroids from field isolates of citrus with exocortis. Citrus viroids were characterized by the physical parameters of electrophoretic mobility, chromatography on CF-11 cellulose and hybridization to cDNA probes of the well characterized citrus viroids, citrus exocortis viroid, CV-Ib from the ‘citron variable viroid’ isolate, and citrus cachexia viroid. These characteristic properties combined with biological distinctions in the host range and symptom expression suggested a scheme for the organization of the citrus viroids into five major groups. The association of the symptoms induced by these citrus viroids in citron cv. Etrog, their organization into individual viroid groups and their presumed relationship to the exocortis disease of citrus are discussed.
Antisera specific for the non-structural proteins 1a and 2a of brome mosaic virus (BMV) were prepared by the gene fusion method. Plasmids were constructed which expressed parts of 1a and 2a in Escherichia coli as fusion proteins with Protein A. After induction of fusion protein synthesis in E. coli, the fusion proteins accumulated in insoluble fractions. Antisera raised against these proteins (anti-1a and anti-2a sera) reacted specifically with the respective proteins translated in vitro and in vivo. These antisera were used to investigate the involvement of the 1a and 2a proteins in the BMV replicase preparation which initiated complementary strand synthesis de novo on BMV RNA. Immunoblot analysis using these antisera revealed the presence of 1a and 2a in the BMV replicase fraction. The replicase activity was inhibited by the addition of anti-1a serum, but not anti-2a serum. Further addition of Protein A-Sepharose to remove each immunocomplex gave similar results. These results suggested the involvement of at least the 1a protein in our BMV replicase preparation.
The replication of cymbidium ringspot tombusvirus satellite RNA was investigated in Nicotiana clevelandii plants. The temperature at which host plants were grown strongly influenced the synthesis of the satellite RNA; high temperatures were detrimental to, or even inhibited, its multiplication. The most favourable temperature range was 16 to 25 °C. The termini of the satellite RNA were studied and a free hydroxyl group was found at the 3′ but not at the 5′ end, which may explain why circular forms were not detected in infected tissue. On the other hand, multimers of double-stranded RNA were present, which may have arisen independently of the formation of circular molecules.