The effect of multiple dispersed copies of the origin-of-assembly sequence from TMV RNA on the morphology of pseudovirus particles assembled in vitro.

Supercoiled plasmid DNA was used as a template to transcribe long concatameric RNA molecules containing several dispersed copies of the origin-of-assembly sequence (OAS) from tobacco mosaic virus (TMV) RNA. When incubated with TMV coat protein "disks" in vitro, these RNAs spontaneously assembled into TMV-like pseudovirus particles. However, as each OAS initiated assembly more or less simultaneously, the concatameric RNA species generated complex nucleoprotein structures of predictable morphology. Similar structures were proposed some time ago (M. E. Taliansky, I. B. Kaplan, L. V. Yarvekulg, T. I. Atabekova, A. A. Agranovsky, and J. G. Atabekov, 1982, Virology 118, 309-316) to account for the RNase-sensitive phenotype of a ts mutant of TMV, Ni2519. These results extend the utility of our RNA-packaging vector system and confirm many of the predictions based on the current model for the self-assembly of TMV.

Evidence that tobacco mosaic virus particles disassemble contranslationally in vivo.

The possibility that a cotranslational disassembly mechanism, similar to that observed when pH 8-treated tobacco mosaic virus (TMV) particles are incubated in an in vitro translation system [T. M. A. Wilson. (1984), Virology 137, 255-265], may be involved in the early stages of virus infection was investigated. Extracts of tobacco leaf epidermal cells, collected between 10 and 70 min after inoculation with 32P- and [3H]leucine-labeled TMV, contained material which had higher buoyant densities in Cs2SO4 gradients and higher 32P:3H ratios than did virus particles. The material sedimented to positions similar to those of in vitro-prepared complexes of partially stripped virus particles and ribosomes and to those of the in vivo-produced complexes of TMV rodlets and in vivo-labeled, nascent polypeptides that formed after inoculation with unlabeled, untreated TMV. In the electron microscope, some of this material resembled the complexes observed in the in vitro translation system. Experiments in which TMV or partially stripped TMV was mixed with epidermal cells from mock-inoculated leaves indicated that the material did not arise by dissociation of virus particles. nor by binding of subcellular components to partially uncoated TMV, during extraction and analysis. These observations provide evidence of the occurrence of a cotranslational disassembly mechanism during the early stages of infection with TMV.

Selective encapsidation of CAT gene transcripts in TMV-infected transgenic tobacco inhibits CAT synthesis.

Young tobacco seedlings (F1-progeny), transformed to express chloramphenicol acetyltransferase (CAT) mRNA with or without a 3'-proximal copy of the origin-of-assembly sequence (OAS) from tobacco mosaic virus (TMV) RNA (residues 5118-5550), were inoculated with TMV. After 21 days, virus symptoms were observed and systemic TMV infections were confirmed by Western blotting for viral coat protein and by electron microscopy of leaf saps. CAT activities were measured in extracts of leaf discs taken before, and 21 days after, virus inoculation. On average, the systemic leaves from TMV-infected CAT-transgenic plants containing the OAS exhibited 3.2-fold less CAT activity than the equivalent leaves from CAT-transgenic control plants lacking the OAS. Hence selective, OAS-dependent encapsidation of nuclear DNA transcripts into TMV-like (pseudovirus) particles can reduce expression of a particular mRNA, post-transcriptionally, in vivo. Furthermore, these data indicate that TMV self-assembly is not restricted to an exclusive subcellular compartment in vivo, and that formation of natural pseudovirions (A. Siegel, Virology 46, 50-59 (1971)) may shut off specific host RNA functions.

Tissue-specific glycogen branching isoenzymes in a multicellular prokaryote, Streptomyces coelicolor A3(2).

In the overtly differentiated colonies of Streptomyces coelicolor A3(2), discrete phases of glycogen synthesis are found at the vegetative/aerial mycelium boundary (phase I) and in the immature spore chains at aerial hyphal tips (phase II). We have characterized two S. coelicolor glgB genes encoding glycogen branching enzyme, which are well separated in the genome. Disruption of glgBl led to the formation of abnormal polyglucan deposits at phase I, with phase II remaining normal, whereas disruption of glgBII interfered specifically with phase II deposits, and not with those of phase I. Thus, each branching enzyme isoform is involved in a different phase of glycogen synthesis. This situation contrasts with that in simple bacteria, which typically have a single set of enzymes for glycogen metabolism, and more closely resembles that in plants.

Purification and preliminary characterization of Spiroplasma fibrils.

Fibrils 3.5 nm in diameter were released from the honeybee spiroplasma (BC3) by treatment with detergents and then purified by isopycnic centrifugation. Purified fibrils were flexuous, of indeterminate length, and had an axial repeat of 8.5 nm. The fibrils were associated in pairs, but in 1 M salt formed aggregates with a marked striated appearance. Pronase completely degraded the fibrils, but trypsin had little effect. The fibrils were composed of a single protein of molecular weight 55,000 which represented about 1% of the total cell protein. A protein of molecular weight 26,000 appeared to be associated with the fibrils. The significance of this in relation to membrane attachment and the possible role of fibrils in maintenance of cell shape and in motility are discussed.

Morphology and ultrastructure of helical and nonhelical strains of Spiroplasma citri.

Cells of the nonhelical strain of Spiroplasma citri underwent changes of morphology comparable to those which occurred in the normal helical strain. Cells of the nonhelical strain had the same ultrastructural features as helical cells and released long flexible fibrils similar to those seen in other spiroplasmas. Nonhelical organisms showed an increased tendency to aggregate, forming cell clusters of an unusual annular form. The cytoplasmic membrane of the nonhelical strain lacked a single protein present in all helical strains. Loss of helicity associated with the senescence of spiroplasma cells was not accompanied by the disappearance of this protein. Differences in colony morphology were shown to be a consequence of motility, and a technique was developed which facilitated the identification of nonmotile organisms.

Functional and evolutionary implications of a survey of various actinomycetes for homologues of two Streptomyces coelicolor sporulation genes.

In Streptomyces coelicolor A3(2) the whiB and whiG genes are essential for sporulation, their deduced products being a possible transcriptional activator and an RNA polymerase sigma factor, respectively. In a survey of DNA from diverse actinomycetes by Southern blotting, all samples tested hybridized with whiB, but only those representing genera capable of producing sporulating aerial mycelium hybridized with whiG. It is postulated that whiB may play a more intimate role in hyphal fragmentation processes (including sporulation) than whiG. The whiB and whiG homologues (whiB-Stv and whiG-Stv) of Streptoverticillium griseocarneum were cloned and sequenced, and subjected to functional tests in S. coelicolor whiB and whiG mutants. The genes were closely similar, but not identical, to their S. coelicolor counterparts at the DNA and deduced protein levels, and both Stv. griseocarnum gene products could function well in S. coelicolor. However, studies with hybrid transcription units suggested that the promoter region of whiB-Stv is somewhat inefficient in S. coelicolor.

Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro.

Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U-32P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2.5% of the RNA (160 5' nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5' oligonucleotide marker (omega) recovered remained constant throughout a 15 min period in SDS. Additional RNase T1-sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions 'destabilized' by ultracentrifugation at between pH 7.2 and 9.2 were quantified using L-[35S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5' leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7.5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that omega at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7.2 to 9.2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3' to, the first AUG codon. We propose that the complete 5' leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly.

The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B. subtilis.

In the mycelial prokaryote S. coelicolor, whiG is a gene dispensable for growth but needed for the earliest stages of spore formation in aerial hyphae. Nucleotide sequencing indicates that whiG encodes an RNA polymerase sigma factor highly similar to the motility sigma factor (sigma 28) of B. subtilis. High copy number of an intact whiG gene caused sporulation in vegetative hyphae that are usually fated to lyse without sporulating. However, the introduction of many copies of a sigma 28-dependent promoter from B. subtilis into S. coelicolor reduced sporulation, suggesting partial sequestration of the whiG gene product by the foreign promoter sequences. We propose that the level of whiG sigma factor is crucial in determining the developmental fate of hyphae.

Deletion of DNA lying close to the glkA locus induces ectopic sporulation in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) J1668 sporulated ectopically in the substrate hyphae (the Esp phenotype) with the same time course as sporulation in the aerial hyphae. Examination of related strains implied that the Esp phenotype was caused by the deletion of DNA that lies close to, but is distinct from, the glucose kinase gene (glkA). Co-transduction of the Esp phenotype with the deletion present in J1668 confirmed this hypothesis. The size of the deletion was found to be 7.4 kb. Construction of a strain carrying both the J1668 deletion and a whiG mutation demonstrated that the Esp phenotype depends on at least one of the genes required for the differentiation of aerial hyphae into spores.

Immunogold localization of the intracellular sites of structural and nonstructural tobacco mosaic virus proteins.

Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic virus (TMV) RNA or the viral coat protein have been used to localize these proteins within virus-infected tobacco leaf cells by an immunogold labeling technique. A protocol is given for low-temperature fixation to facilitate immunogold labeling. In cells of TMV-infected leaf tissue, the 126K protein immunogold label was found almost exclusively in "viroplasms" in the cytoplasm and in pockets of virus particles at the viroplasmic periphery. When utilizing the coat protein antiserum, very little labeling was seen within the viroplasms, although virus particles throughout the cytoplasm were heavily labeled. Viroplasms contained electron-dense rope-like structures embedded in a ribosome-rich matrix. In their "mature" form, viroplasms are the well-known "X body" inclusions. The rope-like structures were up to 1.2 micron long and appear twisted, undergoing several revolutions throughout their length, but were not of a constant pitch. In transverse section, they appeared to be composed of several hollow, radially segmented cylinders 21 nm in diameter, with a 9-nm hole. Antibody labeling showed them to be composed, at least in part, of the 126K protein. Clusters of virus particles at the edge of or within the viroplasms were also labeled with the 126K antiserum, in contrast to virus particles in other areas of the cell, which were not. TMV-infected tobacco mesophyll protoplasts cultured for up to 27 hr did not contain the rope-like ribbons. Instead, isolated protoplasts contained amorphous cytoplasmic areas which were labeled with 126K antibody. Since the 126K protein is most probably a constituent of the TMV RNA-replicating enzyme (replicase), its intracellular location is considered to be indicative of the site of replication of TMV RNA. Therefore these results suggest that replication occurs at the edges of the viroplasms.