T.M.A. Wilson

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.

COMPLEMENTATION OF COAT PROTEIN-DEFECTIVE TMV MUTANTS IN TRANSGENIC TOBACCO PLANTS EXPRESSING TMV COAT PROTEIN

Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) which express tobacco mosaic virus (TMV) U1 strain coat protein (CP) can complement both the assembly and the long-distance spread of CP-defective (DT1) or coat proteinless (DT1G) mutants of TMV. Both mutants arose spontaneously from PM2 and exist only as unencapsidated RNA in the inoculated leaves of control tobacco plants, where they are unable to form virus particles or to spread systemically. TMV CP expressed in transgenic tobacco plants [CP+ line 3404; P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, 1986, Science 232, 738-743] was able to package some of either mutant viral RNA into TMV-like particles in vivo and resulted in the long-range spread of infection. In vivo encapsidated DT1 RNA was recovered and reinoculated onto control or new CP+ transgenic tobacco plants. Localized infection of control plants confirmed that no RNA recombination or reversion of the mutant RNA to wild-type had occurred during passage in the first CP+ plant. In contrast, encapsidated DT1 RNA was unable to produce even local infection in CP+ transgenic plants confirming that CP-mediated protection operates during the early stages of virus infection, including particle uncoating. By positive complementation, these results also confirm that TMV CP is required for the long-distance spread of infection.

Swelling of isometric and of bacilliform plant virus nucleocapsids is required for virus-specific protein synthesis in vitro.

Encapsidated RNA of cowpea chlorotic mottle virus (CCMV), brome mosaic virus (BMV), alfalfa mosaic virus, and southern bean mosaic virus can act as templates for protein synthesis when preswollen virions are added to a wheat germ extract, or when unswollen virions swell in the extract. Encapsidated RNA of turnip yellow mosaic virus, whose particles are unlikely to swell under the conditions used here, did not act as a template. CCMV and BMV, swollen in the extract, gave relatively more RNA 1 and 2 products than when preswollen by dialysis.

Southern bean mosaic virus-specific proteins are synthesized in an in vitro system supplemented with intact, treated virions

RNA encapsidated in icosahedral particles of southern bean mosaic virus (SBMV) can act as a template for protein synthesis in an mRNA-dependent rabbit reticulocyte cell-free translation system, following dialysis of virions against mildly alkaline buffers. Exposure of the SBMV RNA template occurs only after addition of virus particles to the translation system and appears not to involve complete disruption of the protective SBMV capsid.

The kinetics of swelling of southern bean mosaic virus: a study using photon correlation spectroscopy

Southern bean mosaic virus swells upon removal of Ca2+ at pH 8.25. Virions do not seem to aggregate significantly; the z-average hydrodynamic diameter increases from 29.9 nm to 44.0 nm. Swelling is virtually complete within 3 min, and swollen virions have a z-average hydrodynamic diameter similar to that of virions swollen by dialysis overnight.

Southern bean mosaic virus RNA remains associated with swollen virions during translation in wheat germ cell-free extracts

L-[35S]Methionine-labeled translation complexes were prepared by incubating either swollen intact southern bean mosaic virus (SBMV) particles or unencapsidated SBMV RNA in a wheat germ extract. The complexes were analysed by sucrose gradient centrifugation and by electron microscopy and dot blot hybridization of fractions from these gradients. In these complexes, 80 S ribosomes appeared to be associated with intact or near intact particles, suggesting that SBMV particles disassemble only after their RNA has initiated translation. This is in contrast to some other isometric viruses, such as turnip yellow mosaic virus, which appear to release their RNA rapidly prior to translation.

The effect of extraction protocol on the yield, purity, and translation products of RNA from an isometric plant virus

Abstract This is a comparative study of methods used to extract RNA from southern bean mosaic virus (SBMV) for translation in vitro. The methods were: removal of protein, either by 4 M sodium perchlorate or by shaking dissociated virus with a mixture of phenol and chloroform; precipitation of RNA from 2 M lithium chloride; or separation of RNA from protein by sucrose density gradient centrifugation. These methods gave RNA samples of different yields and purities. On translation in vitro the RNA samples stimulated protein synthesis to different extents, and gave different sets of polypeptide products. The best method for preparing SBMV RNA for in vitro translation was that which used the phenol-chloroform mixture.