Spherical Plant Viruses Research Articles

Studies on the assembly of a spherical plant virus. I. States of aggregation of the isolated protein.

The aggregates formed at equilibrium by purified protein from cowpea chlorotic mottle virus have been characterized on the basis of their sedimentation behaviour and appearance in the electron microscope. Between pH 3.5 and 7.5, at ionic strengths greater than 0.2, most of the protein is found in aggregates sedimenting at either 3 S or 50 S. The 50 S aggregate is identified as the reassembled capsid of cowpea ehlorotic mottle virus. Decreasing the ionic strength favours the formation of multi-shelled particles. Below pH 5.5 single- and multishelled particles predominate, while above this pH most of the protein sediments at 3 S. Varying the temperature from 5 °C to 20 °C has little effect on the equilibrium proportions of aggregates although some real differences can be detected. Ionic strength is not as important a variable as pH in determining which protein forms are present (but increasing ionic strength does result in a steady decrease in the proportion of protein in the multi-layer aggregates). The dependence of the equilibrium upon protein concentration shows that capsid formation is a quasi-crystallization: beyond a certain total protein concentration the concentration of 3 S aggregate remains at this “critical” concentration and all further protein goes into 50 S capsid. In addition to shells and variations upon shells, tubes and hexagonal nets of protein subunits have occasionally been seen with the electron microscope.

Dissociation and reassociation of infectious poliovirus particles.

THE first reconstitution of an infectious virion was achieved when Fraenkel-Conrat and Williams1 obtained the typical rods of tobacco mosaic virus (TMV) from its components, RNA and protein. Later, the conditions for the “self-assembly” of TMV were improved so that up to 50% of the viral RNA could be coated with the protein. The reconstituted TMV was shown to be infectious and indistinguishable from native virions by several criteria2. Recent studies revealed that the reconstitution of TMV is a highly specific multi-step procedure, beginning at the 5′-end of the TMV-RNA3–5. In the past five years a number of spherical plant viruses have also been reconstituted6. In experiments with small RNA-bacteriophages very low efficiencies of reconstitution in the range of 10–8 to 10–7 p.f.u. (plaque forming units) per input molecule of RNA were obtained7,8. Reconstitution was improved by the addition of a minor viral protein, the A-protein, to the mixture of RNA and the structural protein. Even so the efficiency of conversion of RNA into infectious particles was in the range of 2×10–6 (refs. 9 and 10). We have reported the first successful restoration of poliovirus infectivity lost on dissociation of the virion by urea-mercapto-ethanol treatment11. Here we present evidence for reconstitution of infectious poliovirus particles from a mixture of poliovirus RNA and polypeptides.

The Multicomponent Nature of Broad Bean Mottle Virus and its Nucleic Acid

Broad bean mottle virus (BBMV) is a spherical plant virus belonging to the same group as brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) (Bancroft, 1970). BMV and CCMV have recently been shown to have divided genomes (Lane & Kaesberg, 1971; Bancroft, 1971) contained in virus particles which sediment as a single species. The RNA from BBMV, when examined in the ultracentrifuge, has been shown to be heterogeneous (Kodama & Bancroft, 1964), consisting of a 17S infectious species, and a 10S species (Bancroft et al. 1968) which was considered to be a degradation product about one-third the size of the large piece. As with the other members of this group, further heterogeneity of the fastest sedimenting species has been found on electrophoresis of the RNA on polyacrylamide gels (Fowlks & Young, 1970).

Uncoating of two spherical plant viruses

Chinese cabbage leaves were inoculated with 32P-labeled TYMV, and barley leaves with 32P-labeled BMV. At various times after inoculation, the proportion of uncoated viral RNA was estimated. In both cases, the uncoating process takes place very soon after inoculation. With TYMV it has been observed that both the rate of uncoating and its time course are very strongly dependent on the ionic strength of the inoculum. Most of the irreversibly bound particles seem able to be uncoated within the first 10 min. Analysis of the proteinaceous compounds formed during the uncoating of RNA showed that empty shells are formed in the case of TYMV, whereas only low molecular weight products occur with BMV.

The effect of an arginyl to a cysteinyl replacement on the uncoating behaviour of a spherical plant virus

A mutant of cowpea chlorotic mottle virus is described which, when extracted in the absence of cysteine and diethyldithiocarbamate, is noninfective. The mutant virus apparently cannot be uncoated to initiate infection because phenol extracts of its RNA are infective. The mutant is more pH and salt stable than is the wild-type virus. The properties of the mutant are related to the replacement of an arginyl by a cysteinyl residue.

Structures derived from cowpea chlorotic mottle and brome mosaic virus protein

The behavior at different salt and acidic pH levels of protein isolated from three spherical plant viruses was investigated. Broad bean mottle virus protein did not aggregate into recognizable organized structures. Protein from brome mosaic virus formed capsids similar to those of the virus at a pH and salt-dependent efficiency with best yields at pH 5.0 in 0.2 M NaCl. Best yields for cowpea chlorotic mottle virus protein capsids occurred at pH 5.0 in 0.1 M NaCl or from pH 3.5 to 5.0 in 0.2 M NaCl. Protein from the latter virus also formed double-shelled and rosettelike particles in 0.2 M NaCl from pH 5.3 to 5.7. Narrow tubes were made at pH levels of 6.0 and higher, and the tubes were accompanied by T = 1 and T = 3 particles if ribonu-clease-digested RNA was present. In the absence of NaCl in 0.01 M acetate buffer at pH 4.0 and 4.5, laminar and platelike aggregates were found with CCMV protein. The production and some of the properties of the various forms are described.

Studies on the bacteriophage MS2: III. Sedimentation heterogeneity of viral RNA preparations

Some RNA preparations, isolated from the bacteriophage MS2, gave rise upon analytical ultracentrifugation to several sharp boundaries in addition to the boundary corresponding to the intact molecular species (27 S). Most often a tripartite distribution was observed, containing the 27 S component, a second component (S 2) moving at 21 S, and a third component (S 3) moving at 15 S. It is concluded that the smaller components are formed by nonrandom fragmentation. The 21 S component and the 15 S component may correspond to a half-molecule and a quarter molecule, respectively, or more likely to a two-third and a one-third piece. Sometimes, a 38 S dimer species was observed. The tripartite pattern is strongly reminiscent of similar sedimentation heterogeneity observed for the RNA of several spherical plant viruses and perhaps animal viruses. It is possible that all these RNA's have in common a specific, perhaps function-related, tertiary structure.

A Rapid Method to Detect Particles of Some Spherical Plant Viruses in Fresh Preparations

A Rapid Method to Detect Particles of Some Spherical Plant Viruses in Fresh Preparations Get access E. W. KITAJIMA E. W. KITAJIMA Virus Department, Instituteo AgronomicoCampinas, S.P. Brazil. Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Electron Microscopy, Volume 14, Issue 2, 1965, Pages 119–121, https://doi.org/10.1093/oxfordjournals.jmicro.a049473 Published: 01 January 1965 Article history Published: 01 January 1965 Received: 19 May 1965

REMOVAL OF HOST ANTIGENS FROM PLANT VIRUS PREPARATIONS BY ION EXCHANGE CHROMATOGRAPHY

The separation of a carnation virus from smaller cowpea-host antigens was achieved by column chromatography on a strongly basic ion-exchange resin. The separation was demonstrated by infectivity and serological tests, analytical ultracentrifugation, and ultraviolet spectrophotometry. The method is proposed for the removal of similar host components from preparations of other spherical plant viruses.

Bromegrass Mosaic Virus: a Virus containing an Unusually Small Ribonucleic Acid

CHEMICAL and physical properties of viruses are being investigated in several laboratories in order to obtain structural information which relates to questions of virus synthesis and genetics. Bromegrass mosaic virus, a spherical plant virus, may qualify as a useful test object for such investigations. Data reported here show that this virus is particularly small, and furthermore that the molecular weight of its ribonucleic acid is much smaller than that of any other known virus.

The symmetries of the protein and nucleic acid in turnip yellow mosaic virus: X-ray diffraction studies

Turnip yellow mosiac virus (TYMV) is an approximately spherical plant virus having a (hydrated) spherically-averaged diameter of 280 A and containing about 60% protein and 40% RNA (Markham, 1951). The protein is in the form of a spherical shell with the RNA contained inside. Associated with the virus is the “top component” which consists of empty protein shells containing no RNA. Previous X-ray diffraction studies (Klug, Finch & Franklin, 1957) of crystals of TYMV have shown that the virus particles have the symmetry of the tetrahedral point group (23), with a very strong tendency towards icosahedral (532) symmetry. It was suggested that the icosahedral symmetry was due to the protein component. We have now extended these studies on the virus crystals to higher angles of diffraction. Our previous observations are confirmed and it is shown that some part of the virus particle has 532 symmetry developed to a resolution of at least 4 A. We have now also obtained photographs of a single crystal of the top component and these confirm that the protein shell has 532 symmetry. It is therefore made up of 60 asymmetric (structurally equivalent) units so that the number of chemical units of any one kind must be a multiple of 60. Recent electron microscope observations presented in the accompanying papers (Huxley & Zubay, 1960; Nixon & Gibbs, 1960), show that the virus is composed of 32 protuberances arranged so that 20 fall at the vertices of a pentagonal dodecahedron and 12 at those of an icosahedron. The relation between the X-ray and the electron microscope results and the available chemical evidence is discussed. All the present data can be reconciled if the protein shell is made up of 180 (or, less likely, 120) chemical subunits arranged regularly in 20 groups of 6 (or 3) at the dodecahedral vertices and in 12 groups of 5 at the icosahedral vertices, but other, only statistically regular, models cannot be ruled out. Differences are observed between the X-ray diffraction patterns of crystals of the virus and of the top component. These must be due directly or indirectly to the RNA, and show the RNA to possess, at least in part, some kind of regular structure. The differences observed are consistent with a configuration having 23 symmetry, which, if correct, would imply that the RNA is made up of 12 geometrical subunits. This could still be reconciled with a single chemical chain. The problem of packing such a chain into a spherical particle with cubic symmetry is discussed. However, the apparent 23 symmetry of the RNA may arise as a statistical average over the many virus particles in a crystal, the RNA in a single particle having lesser symmetry. The various possibilities are discussed.

The structure of the protein shell of turnip yellow mosaic virus

Turnip yellow mosaic virus (TYMV) is an approximately spherical plant virus, having a diameter of 280 to 300 A and containing 60% protein and 40% RNA; the protein is believed to form a spherical shell around a nucleic acid core. It has been shown previously by X-ray diffraction that the virus particles must have cubic symmetry, and it has been argued on theoretical grounds that viruses having cubic symmetry and containing a spherical shell of protein were likely to have that protein shell built up of subunits arranged in a polyhedron having either tetrahedral (23), octahedral (432) or icosahedral (532) symmetry elements. We have examined TYMV in the electron microscope by the negative staining method, and have found that regularly disposed subunits may be discerned on the surface of the particles. There appear to be 32 such subunits, arranged on or near the vertices of either a pentakis dodecahedron or a rhombic triacontahedron (or other polyhedra approximating to those forms). These are semi-regular poly-hedra having 532 symmetry, the former having 60 identical triangular faces and the latter 30 identical rhombic faces; both have 32 vertices, which fall into two separate groups, 12 vertices lying on fivefold axes and 20 vertices lying on threefold axes. Thus the subunits occupy two different types of position, and can be of two different species. Because the subunits lie on rotation axes, they must contain the appropriate number of identical structural units. These results agree with the suggestion made on the basis of the X-ray results that the particles have 532 symmetry, but they conflict with the more tentative suggestion that the particular polyhedron involved contained 60 subunits placed at the vertices of a snub dodecahedron. A possible way in which the X-ray results can be reconciled with the present observations is suggested.