T. Hatta

Studies on encapsidated viroid-like RNA I. Characterization of velvet tobacco mottle virus

Velvet tobacco mottle virus (VTMoV) isolated from Nicotiana velutina growing wild in arid Central Australia was transmitted by inoculation to a limited number of plant species of which N. clevelandii was the most convenient experimental host. The virus was also transmitted from field-grown plants toN. velutina and N. clevelandii by the mirid Cyropeltis nicotianae. VTMoV preparations purified by clarification with organic solvents and differential centrifugation contained polyhedral particles about 30 nm in diameter sedimenting as a single component at about 115 S. The particles were shown to be located in the nucleus, cytoplasm, and vacuoles of infected plant cells. Virus dissociated in the presence of mercaptoethanol and sodium dodecyl sulfate (SDS) separated into one major and two minor polypeptides with estimated molecular weights of 33,000, 36,000 and 31,000, respectively. Single-stranded RNA isolated from VTMoV by extraction with phenol was separated into five components with apparent molecular weights of 1.5 x 10(6), 0.63 x 10(6), 0.25 x 10(6), 0.16 x 10(6), and 0.12 x 10(6) referred to as RNAs 1, 1a, 1b, 2, and 3, respectively. It appears that RNAs 1a and 1b are breakdown products of RNA 1, as shown elsewhere, and electron microscopic examination of the other species showed that whereas RNAs 1 and 3 are linear molecules, RNA 2 is circular. The similarity of RNAs 2 and 3 to the RNA of viroids is discussed. VTMoV has been compared with several RNA plant viruses with small polyhedral particles. Only solanum nodiflorum mottle virus appears to share some of its unique features and the two have been shown to be antigenically related.

Circularity of the ribonucleic acids associated with cadang-cadang disease

The ribonucleic acids, ccRNA-1 and ceRNA-2, associated with cadang-cadang disease are circular single-stranded molecules comprising 310 +/- 3 nucleotides and 438 +/- 5 nucleotides, respectively; their molecular weights are estimated to be 1.05 and 1.49 x 10(5) daltons. Therefore ceRNA-2 appears not to be a dimer of ccRNA-1, although it is known to have nucleotide sequences in common with ccRNA-1. Differences in the native structure of the two RNAs as determined by length measurements may account for previously described differences in their properties. Both RNAs resemble viroids, but ccRNA-1 is smaller, and ceRNA-2 is larger, than the viroids which have been characterized.

The bipartite genome of red clover necrotic mosaic virus

Purified preparations of red clover necrotic mosaic virus isolated in Australia have been shown to contain three RNA components whose electrophoretic mobilities in polyacrylamide gel electrophoresis indicate molecular weights of 1.5 x 106 (RNA 1), 0.5 x 10(6) (RNA 2), and 0.14 x 10(6) (RNA 3). Comparisons of the RNAs by hybridization analysis with 3H-labeled complementary DNAs synthesized in vitro have established that RNAs 1 and 2 are unique RNA molecular species with little or no sequence homology between them. However, RNA 3 appears to be a complex mixture of breakdown fragments of both RNA 1 and RNA 2. Infectivity experiments with highly purified preparations of RNAs 1 and 2 have demonstrated that both molecules are essential for infectivity.

The composition of Chloris striate mosaic virus, a geminivirus.

Preparations of Chloris striate mosaic virus (CSMV) were shown to contain circular single-stranded DNA with a molecular weight of 7.1 x 10(5) and a polypeptide molecular weight of 92.79 +/- 0.06 x 10(4). The buoyant density in CsCl of intact CSMV particles was shown to be 1.35 g.cm(-3). From these data and the capsomeric structure of CSMV (T. Hatta and R. 1. B. Francki, 1979, Virology92, 428-435) it is concluded that each geminate particle of CSMV has a molecular weight of about 3.8 x 10(6) and contains a single molecule of ss-DNA.

Studies on encapsidated viroid-like RNA. III. Comparative studies on RNAs isolated from velvet tobacco mottle virus and Solanum nodiflorum mottle virus.

Solanum nodiforum mottle virus (SNMV) encapsidates two unique RNA species as shown by 3H-complementary DNA hybridization analysis. They are RNA 1, a long linear single-stranded molecule with a molecular weight of 1.5 x 10(6) and RNA 2 and RNA 3 which are circular and linear forms of the same molecule, respectively. Electron microscopic examination and thermal denaturation studies indicate a viroid-like structure for RNA 2 of SNMV. Electrophoretic analysis of the viroid-like RNA encapsidated by SNMV indicates it has a slightly larger molecular weight (0.13 x 108) than the viroid-like RNA recently isolated from velvet tobacco mottle virus (VTMoV). Complementary DNA (cDNA) analysis revealed that SNMV RNA 2 contains the entire nucleotide sequence of VTMoV RNA 2, plus an additional 40-50 nucleotides not present in the latter. Comparative sequence homology studies between the large RNAs encapsidated by SNMV and VTMoV (both of MW 1.5 x 10(6)) revealed only partial homology (20%). However, the level of S1 nuclease resistance could be increased by the presence of higher salt concentrations during hybridization and subsequent S1 nuclease assay. This indicates that many smaller regions of homology are present between VTMoV RNA 1 and SNMV RNA 1. This is the second example of an encapsidated viroid-like RNA.

The fine structure of chloris striate mosaic virus.

Abstract Geminate particles measuring about 18 × 30 nm in negatively stained or freeze-dried and shadowed preparations of chloris striate mosaic virus (CSMV) have been examined by high resolution electron microscopy. Each particle appears to be constructed from two incomplete icosahedra with T = 1 surface lattice having a total of 22 capsomeres. Some virus particles analyzed at various angles in an electron microscope equipped with a goniometer stage occurred as geminate structures in situ. We conclude that the morphology of native CSMV particles is geminate.

Enzyme cytochemical identification of single-stranded and double-stranded RNAs in virus-infected plant and insect cells.

Abstract A method is described for identifying and locating single-stranded (ss) and double-stranded (ds) RNAs by electron microscopy in plant and insect cells. Single-stranded RNA in aldehyde-fixed tissues is identified by its susceptibility to pancreatic ribonuclease when incubated in media containing both low or high salt concentrations, and dsRNA is identified by its susceptibility to the enzyme in media of low but not high salt concentrations. The validity of this method for identification of intracellular dsRNA has been verified with plant and insect cells infected with Fiji disease and maize wallaby ear viruses, respectively, both of which contain dsRNA genomes. The method has also been used for the identification of dsRNA in vesicles of virus-induced inclusions in plant cells infected with Echtes Ackerbohnenmosaikvirus, a member of the comovirus group which contains an ssRNA genome. The need for critical interpretation of observations of structures in RNase-treated cells is discussed.

Morphology of fiji disease virus

Abstract Polyhedral particles in crude extracts and partially purified preparations from leaf gall tissue of Fiji disease virus (FDV)-infected sugarcane have been examined in an electron microscope after negative staining or freeze-drying and shadowing. The FDV particle is a double-shelled, icosahedral structure consisting of a stable core surrounded by an unstable outer shell. The intact virus particle in freeze-dried and shadowed preparations is about 67 nm in diameter with projections (A spikes), about 14 nm wide and 8 nm long, at the 12 vertices of the icosahedron. The A spikes are attached to the projections (B spikes) of the 54-nm core which are about 19 nm wide and 9 nm long. The resolution of the FDV-particle substructure in negatively stained preparations was not sufficient to determine the size and arrangement of the capsomeres. The morphology of FDV was compared to that of reovirus prepared for electron microscopy by similar techniques; although the two viruses possess similar general structure and complexity, some significant differences were readily recognized. It is concluded that the FDV particle is remarkably similar to that of maize rough dwarf virus, confirming the suggestion that the two viruses are closely related.

Purification and some properties of reovirus-like particles from leafhoppers and their possible involvement in wallaby ear disease of maize

Reovirus-like particles, occurring in association with viroplasms, crystalline arrays and tubules, in the cytoplasm of Cicadulina bimaculata capable of inducing wallaby ear disease in maize, were purified from the insects by differential centrifugation, treatment with the nonionic detergent, Nonidet P-40, and sucrose density gradient centrifugation. The purified particles have a double-shelled icosahedral structure about 70 nm in diameter with external projections (A spikes) about 10 nm long located at the 12 vertices. These intact particles (IPs) are morphologically similar to those of Fiji disease virus (FDV), but are more stable. Cores were produced by enzymatic digestion of IPs with alpha-chymotrypsin. The cores are icosahedra about 57 nm in diameter with projections (B spikes) located at the 12 vertices, resembling those of FDV and cytoplasmic polyhedrosis virus. Immunization of a rabbit with purified IPs resulted in the production of antibodies specific to IPs, cores, and dsRNA. Immunoelectron microscopic investigations revealed that there is no relationship between this virus and FDV, maize rough dwarf, oat sterile dwarf, pangola stunt, and rice ragged stunt viruses, all members of the genus Fijivirus in the family Reoviridae. The nucleic acid extracted from partially purified virus was resolved into 10 segments by polyacrylamide gel electrophoresis. Reovirus-like particles or viroplasms could not be detected in thin sections of maize seedlings colonized by C. bimaculata showing wallaby ear symptoms. In the light of these data the possible etiology of wallaby ear disease is discussed.

Structure of the antigen from Fiji disease virus particles eliciting antibodies specific to double-stranded polyribonucleotides.

Antisera with Fiji disease virus (FDV) protein-specific and double-stranded (ds) RNA-specific antibodies were prepared by injecting rabbits with highly purified preparations of FDV cores. Electron microscopic examination of negatively stained core preparations disclosed the presence of icosahedral particles, about 54 nm in diameter, with projections (B spikes) at their vertices as previously reported. However, observations on similar preparations by electron microscopy of specimens shadowed at a low angle, by immune electron microscopy and by examination of thin sections of pelleted material, established that the particles consisted of cores to which some ds RNA strands were attached externally. The relationship of FDV core structure to its immunogenic properties is discussed.