- Volume 70, Issue 10, 1989

An organelle-free fraction with the ability to synthesize citrus exocortis viroid (CEV) RNA was prepared from nuclei-rich samples taken from CEV-infected Gynura aurantiaca D.C. leaf tissue. This extraction was accomplished in the presence of the detergent sarkosyl. Characterization of the viroid synthetic reaction demonstrated that the solubilized complex retained the properties displayed by intact nuclei. These include optima of 1 mm-MnCl2, 10 mm-MgCl2 and 25 mm-(NH4)2SO4, as well as inhibition by α-amanitin (90% at 4 μg/ml) and by the chelators o-phenanthroline (78% at 5 mm) and hydroxyquinoline (45% at 5 mm). Nucleic acid-binding agents such as ethidium bromide and actinomycin D showed a low, non-specific inhibition at relatively high concentrations. Sucrose gradient sedimentation analysis of the sarkosyl supernatant (SSN) components before and after the RNA synthesis showed that the bulk of the progeny remained associated with the viroid complex which sedimented faster than phenol-treated viroid RNA. A high percentage of the CEV RNA molecules migrated in electrophoresis as the circular form. This suggests that all elements necessary for the synthesis of viroid RNA and processing to circular structures are present in the SSN. This endorses the potential of these subnuclear preparations for the study of the processes involved in viroid replication.

Amino acid sequences of 16 geminivirus replication-associated proteins and 15 coat proteins were aligned and a new computer program was used to calculate the minimum mutation distances for all possible pairwise comparisons. These data were used to construct phylogenetic trees. Trees based on coat proteins had two main branches which were positively correlated with vector specificities of the viruses. Trees based on replication-associated proteins also had two main branches which were positively correlated with viral host specificities for either monocotyledonous or dicotyledonous plants. Therefore, evolutionary pressures on coat proteins and replication-associated proteins are probably highly influenced by vectors and hosts, respectively. Geminiviruses that infect dicotyledonous plants may be divided further by geographical origins into Old World and New World viruses. These results suggest the possible geographical origins of some geminiviruses, that new taxa should be erected, and have implications for distinguishing viruses and strains.

Ultrastructural examination of leaf tissue of Nicotiana benthamiana infected with Indian cassava mosaic virus (ICMV) revealed abnormalities in phloem and, occasionally, xylem cells. Nuclei contained granular inclusion bodies which seemed to be largely composed of virus-like particles and were shown by immunogold labelling (IGL) to be rich in ICMV coat protein. Later in infection, hollow spheres made up of fibrillar material were produced. Virus coat protein was not a necessary component of these structures but was sometimes found in their hollow centres. Cytoplasmic abnormalities were uncommon but a few vascular parenchyma cells contained paracrystalline aggregates which seemed to be made up of hollow tubes 30 to 40 nm in diameter. Some of these same cells also had large areas of cytoplasm which contained numerous randomly orientated tubules about 20 nm in diameter. No ICMV coat protein could be detected by IGL in either the paracrystalline or the tubule-containing inclusions. The ultrastructural effects of ICMV resemble those of other whitefly-transmitted geminiviruses, but with some differences of detail, and they also include types of abnormality not previously recorded.

The distribution of parsnip yellow fleck virus particle antigen in infected cells of Nicotiana clevelandii or Spinacia oleracea was examined by immunogold labelling of ultrathin sections. Best results were obtained by pretreating sections with Decon 75 followed by long incubation times on antiserum (16 h) and gold probe (6 h). The cytoplasmic inclusions induced by infection have three main components: accumulations of 20 to 30 nm diameter tubules, granular bodies and amorphous matrix material. Much gold label was located over the areas of amorphous matrix material whereas the other components of the inclusions were not labelled. Specific but less dense labelling was observed over virus-induced cell wall outgrowths, and over other areas of cell wall and some nuclei in infected cells. Virus-like particles found in 45 nm diameter tubules within the cell wall outgrowths were not labelled, perhaps because they were inaccessible to the antibody. The results indicate that large amounts of virus particle antigen are present in cells. However, the number of recognizable virus particles was considerably less than expected from the amount of virus extracted from leaves.

The possibility of complementation of the cell-to-cell spread (within the inoculated leaf) between different related and unrelated plant viruses has been studied. Various tobamoviruses (tobacco mosaic, sunn-hemp mosaic, cucumber green mottle mosaic viruses and ‘tobamovirus from orchids’) can facilitate each other’s replication in non-permissive hosts or at a temperature non-permissive for transport of one of the virus partners, probably by complementation of transport functions. Complementation of movement also occurred between some, but not all unrelated viruses tested. The complementation in transport function seems to be non-specific: it can occur between viruses even if their putative transport proteins significantly differ in structure. Consequently these viruses were classified tentatively into different ‘transport groups’.

The nucleotide sequence of DNA complementary to the eighth largest (S8) of the 12 genome segments of rice dwarf virus was determined. This genome segment is 1424 nucleotides in length and has a single long open reading frame extending 1260 nucleotides from the first AUG triplet (residues 24 to 26). The predicted translational product comprises 420 amino acids and has an M r of 46422. The amino acid sequences of several peptide fragments of the major outer capsid protein were found to be contained in the predicted translational product of the above nucleotide sequence. This protein, previously reported to be 43K, is encoded by genome segment S8 and therefore renamed the 46K protein.

Complementary DNAs of two different satellite RNA isolates from cucumber mosaic virus (CMV), Y from Japan and Ra from France, have been cloned in a transcription vector containing the Pr promoter. When inoculated on plants with CMV RNA (strain KIN), the transcripts of the cloned Y satellite cDNA elicit a bright yellow mosaic on tobacco and a lethal necrosis on tomato. Addition of the transcripts of the Ra satellite cDNA to an inoculum of CMV RNA resulted in symptom attenuation on both tobacco and tomato, in agreement with the characterized symptoms of the natural satellite. Recombinant molecules involving these two satellites have been constructed in order to determine which parts of the Y satellite RNA are involved in symptom induction. The determinant for symptom production on tobacco lies in the region between nucleotides 1 and 219. The domain for necrotic symptoms on tomato resides on the 3′ half of the molecule beyond nucleotide 219.

The sequence of the 3′ 1106 nucleotides of the watermelon mosaic virus 2 (WMV 2) genome has been determined. The sequence contains the complete coding region of the viral coat protein followed by a 3′ untranslated sequence of 251 nucleotides. When these sequences were compared with the equivalent regions of the N strain of soybean mosaic virus (SMV-N), the coat protein coding regions were 82% homologous, whereas the 3′ untranslated sequences were 78% homologous. Optimal alignment of the 3′ untranslated regions of RNA from 13 strains of seven other distinct potyviruses revealed that the degree of homology between strains was in the range 83 to 99%. In contrast, the sequences from distinct viruses had identities in the range 39 to 53%, comparable to the level of identity found between the 3′ non-coding regions of viruses from unrelated plant virus groups. On the basis of these results, WMV 2 and SMV-N could be regarded as strains of one virus. These results also suggest that the sequence of the 3′ untranslated region of the potyvirus genome may be an accurate marker of genetic relatedness and could serve as an aid to identification and classification of potyviruses.

The infectivity of plum pox potyvirus (PPV) RNA was decreased by treatment with proteases. Ribonuclease digestion of iodinated PPV RNA yielded material which had an electrophoretic mobility corresponding to M r 22000. This protein presumably corresponds to the protease-sensitive structure needed for infectivity. A protein-linked RNase T1-resistant oligonucleotide, 38 nucleotides long, was sequenced and shown to correspond to the 5′ terminus of the RNA by sequence comparison to the RNAs of two other potyviruses, tobacco etch virus and tobacco vein mottling virus. A 12 nucleotide block was found to be completely conserved in the RNAs of the three viruses.