Potato spindle tuber virus: a plant virus with properties of a free nucleic acid.

Infectious entities, extractable, with phosphate buffer, from tissue infected with potato spindle tuber virus and inciting symptoms on tomato that are typical of this virus, have properties incompatible with those of conventional virus particles. The infectious particles sediment in sucrose density gradients at approximately the same rate as particles with a sedimentation coefficient of 10S, are insensitive to treatment with organic solvents, and can be concentrated by ethanol precipitation. Treatment with phenol changes neither their infectivity nor their sedimentation properties. Infectivity is insensitive to deoxyribonuclease, but at low ionic strength it is sensitive to ribonuclease. At high ionic strength, infectivity partially survives incubation with ribonuclease. These properties, as well as elution patterns from columns of methylated serum albumin, suggest that the extractable infectious agent may be a double-stranded RNA.

Protein-RNA interactions in an icosahedral virus at 3.0 A resolution.

Nearly 20 percent of the packaged RNA in bean-pod mottle virus (BPMV) binds to the capsid interior in a symmetric fashion and is clearly visible in the electron density map. The RNA displaying icosahedral symmetry is single-stranded with well-defined polarity and stereochemical properties. Interactions with protein are dominated by nonbonding forces with few specific contacts. The tertiary and quaternary structures of the BPMV capsid proteins are similar to those observed in animal picornaviruses, supporting the close relation between plant comoviruses and animal picornaviruses established by previous biological studies.

Circular dichroism and fluorescence studies on potato virus X and its structural components.

Circular dichroism (DC) measurements of the coat protein subunits of potato virus X show that native subunits that can reassemble with RNA to form infectious virus particles have appreciable alpha-helical structure. The CD of intact potato virus X was less intense below and more intense above 250 nm, and the maxima and minima were at longer wavelengths, than those of a CD spectrum computed from the individual contributions of the coat protein and RNA. The differences between the measured and computed spectra below 250 nm were attributed to the effects of differential light scattering and absorption flattening on measurements of the virus particle CD. The differences at longer wavelengths, were the CD contribution of the nucleic acid predominates, probably reflect the difference between a base-paired conformation of the RNA in solution and the more rigid single-stranded conformation imposed by the structure of the virus. The CD evidence suggests that the tertiary structure and potato virus X coat protein subunits in solution and in intact virus particles is similar. Both CD and fluorescence emission results indicate differences between the tryptophan environment in dissociated protein subunits and that in intact virus. These are attributed to local differences in subunit conformation or to the occurrence of intersubunit interactions involving tryptophan in the intact virus.

Natural fluorescence properties of brome mosaic virus protein.

Some of the fluorescence properties of brome mosaic virus protein in different states of aggregation (dimer, capsid) have been studied, in particular the emission spectra, fluorescence quantum yields and lifetimes, as well as the effect of external quenchers and of temperature on the fluorescence. Brome mosaic virus protein, which contains two tryptophan and five tyrosine residues per monomer, displayed an unusual fluorescence spectrum maximum of 308 nm at pH 7.4 when excited at 280 nm. The emission maximum was shifted to 327 nm when excited at 295 nm. Analysis of the results showed that the tyrosine emission is characterized by a high value for the quantum yield (phi = 0.07), which is consistent with a location of most of these residues in helical regions of the protein, while the tryptophan emission is strongly quenched (phi=0.035). The effects of external quenchers suggested that two kinds of tryptophan residues might exist, one buried (phi=0.056) and one exposed, the quantum yield of the latter being particularly low (phi=0.014). The tryptophan fluorescence quenching is partially removed at pH 8.4 and totally eliminated after chemical (guanidinium chloride) or thermal denaturation of the protein. Formation of capsid induced an additional quenching of the exposed tryptophan residue while interaction with the RNA in the virus did not modify the emission parameters of the protein.

[Determination of the primary structure of alfalfa mosaic virus (strain S) coat protein. II. Complete sequence of the protein (author's transl)]. / Détermination de la structure primaire de la protéine du virus de la mosaïque de la luzerne (souche S). II. Sequence complete de la protéine.

The alfalfa mosaic virus protein was submitted to the action of cyanogen bromide. Four peptides were isolated. Study of these peptides allowed us to determine the order. Then protein was submitted, after S-carboxymethylation or S-aminoethylation, to the action of different proteolytic enzymes: trypsin, chymotrypsin, thermolysin and papain. The peptides issued from these different hydrolysis were separated on Dowex 50 X4 and Dowex 1 X2, and their amino acid composition was determined. The use of classical methods of sequence determination, of mass spectrometry and for one case the use of a sequencer, lead to the obtention of the primary structure of all the tryptic peptides. The studies of chymotryptic, thermolytic and papainic hydrolysates, and of cyanogen bromide rupture, allowed us to isolate the overlapping peptides which were necessary for the reconstitution of the complete proteic chain.

Fluorescent study of tobacco mosaic virus protein.

Protein fluorescence properties of tobacco mosaic virus [3 Trp residues per monomer (positions 17, 52, 152)] and of two tobacco mosaic virus mutants [green tomato atypical mosaic virus, 2 Trp (52, 152) and cucumber virus4, 1 Trp (unknown position)] have been studied. Emission spectra, fluorescence quantum yields and lifetimes were determined. Results showed that protein fluorescence is due to buried Trp only, except for the cucumber virus4 strain, in which Tyr also contributed to the emission. Comparison of the three strains showed that Trp 17 and Trp 52 have high fluorescence yields (phi17 = 0.29; phi52 = 0.37) whereas Trp 152 (probably present in cucumber virus4) is strongly quenched (phi152 = 0.035). An unusually efficient Tyr leads to Trp energy transfer was observed in tobacco mosaic virus protein, indicating that most of four Tyr residues are located near the highly fluorescent Trp.

Structure of RNA in satellite tobacco necrosis virus. A low resolution neutron diffraction study using 1H2O/2H2O solvent contrast variation.

The crystal structure of satellite tobacco necrosis virus has been studied by neutron diffraction at 16 A resolution using the technique of 1H2O/2H2O solvent contrast variation to distinguish between the regions of protein and nucleic acid. The RNA density is essentially localized in a region just inside the protein coat, leading to a significant interaction between the two components. From the appearance of the RNA density we conclude that the protein coat imposes partial icosahedral symmetry on a significant proportion of the nucleic acid. The shape and dimensions of the major part of this density suggests that about 72% of the total RNA could be double-helical in structure. The most important interaction between the two components of the virus occurs between the N-terminal triple-helical arms of the protein subunits and those regions of the RNA density that could have a double-helical secondary structure.

Structure of tomato bushy stunt virus. V. Coat protein sequence determination and its structural implications.

We report the chemically determined sequence of most of the polypeptide chain of the coat protein of tomato bushy stunt virus. Peptide locations have been determined by comparison with the high-resolution electron density map from X-ray crystallographic analysis as well as by conventional chemical overlaps. Three small gaps remain in the 387-residue sequence. Positively charged side-chains are concentrated in the N-terminal part of the polypeptide (the R domain) as well as on inward-facing surfaces of the S domain. There is homology of S-domain sequences with structurally corresponding residues in southern bean mosaic virus.

Crystallization and preliminary X-ray study of a double-shelled spherical virus, rice dwarf virus.

Rice dwarf virus (RDV) is a double-shelled spherical plant virus consisting of 46,000 Mr capsid and 114,000 Mr core proteins and minor structural proteins, and containing 12 genome segments of double-stranded RNA. The virus has been crystallized in the cubic space group I23 with a = 789 A. There are two particles per unit cell, each positioned on a point of 23 symmetry. Packing considerations showed that the diameter of the virus particle is 693 A. The crystals diffract to at least 6.5 A resolution.

Structure of turnip crinkle virus. III. Identification of a unique coat protein dimer.

The minor structural protein (p80), found in about one copy per virion in turnip crinkle virus (TCV), is shown by amino acid analysis and peptide mapping to be a covalent dimer of the major coat protein (p40). The covalent linkage occurs near the N termini of the crosslinked chains. These data suggest that TGV and related viruses contain 178 copies of p40 (89 non-covalent dimers) and one copy of p80 (covalent dimer of two additional p40 chains). The presence of p80 in the salt-stable RNA-protein complex formed when TCV dissociates, as described in an accompanying paper, indicates that the covalent modification affects binding to RNA. We suggest that p80 might be the final dimer to be incorporated into the shell and that it might also be the site for initiation of uncoating.

Structure of satellite tobacco necrosis virus after crystallographic refinement at 2.5 A resolution.

The structure of the protein subunit of satellite tobacco necrosis virus has been solved at 3.7 A resolution. We have now crystallographically refined the original model and extended the resolution ot 2.5 A in order to get a model accurate enough to explain the details of the subunit interactions. The refinement was done with a novel method utilizing the icosahedral symmetry of the virus particle. The final model shows a complicated network of interactions, involving salt linkages, hydrogen bonds and hydrophobic contacts. In addition, we have located three different metal ion sites in the protein shell, linking the protein subunits together. These sites are probably occupied by calcium ions. One site is found in a general position near the icosahedral 3-fold axis of the virus. The ligands form an octahedral arrangement, with two main chain carbonyl oxygens (0-61 and 0-64), one carboxylate oxygen (OD1 from Asp194) of the same subunit and a second carboxylate oxygen (OE1 of Glu25) from a 3-fold related subunit. Two water molecules complete the octahedral arrangement. A second site is on the icosahedral 3-fold axis and is liganded by the carboxylate oxygens of the 3-fold related Asp55 residues. The third metal ion site is found on the 5-fold axis, liganded by the five carbonyl oxygens of Thr138 and two water molecules. We are unable to locate the first 11 N-terminal amino acid residues, which point into the virus interior. No interpretable density for RNA has been found, indicating that the nucleic acid of the virus does not have a unique orientation in the crystal.

Structure and assembly of turnip crinkle virus. I. X-ray crystallographic structure analysis at 3.2 A resolution.

The structure of turnip crinkle virus has been determined at 3.2 A resolution, using the electron density of tomato bushy stunt virus as a starting point for phase refinement by non-crystallographic symmetry. The structures are very closely related, especially in the subunit arm and S domain, where only small insertions and deletions and small co-ordinate shifts relate one chain to another. The P domains, although quite similar in fold, are oriented somewhat differently with respect to the S domains. Understanding of the structure of turnip crinkle virus has been important for analyzing its assembly, as described in an accompanying paper.

Sequential removal of Ca2+ from satellite tobacco necrosis virus. Crystal structure of two EDTA-treated forms.

Two forms of EDTA-treated satellite tobacco necrosis virus (STNV) have been studied with X-ray crystallography methods. The crystals of both forms were isomorphous with native STNV crystals, and (FEDTA-Fnat) maps as well as (2FEDTA-Fnat) maps were calculated with phases from the native structure. The maps were based on partial data sets to 2.8 A resolution, and averaged using the 60-fold non-crystallographic symmetry. In the first crystal form, calcium ions were absent from one of the three sites in the icosahedral protein shell. The crystals were produced at pH 5.0 from a virus solution treated with EDTA at pH 6.5. The virions were not expanded, and no essential changes were seen in the protein shell. In the second crystal form, all calcium ions in the protein shell were absent. The virus material in these crystals had been subjected to treatment with EDTA at pH 8.0 before crystallization at pH 6.5. The high pH treatment caused degradation of the viral RNA. No expansion of the virion had occurred and all protein--protein contacts were retained. These results are compared with the previously presented low-resolution structure of slightly expanded STNV with intact RNA, where calcium ions from two sites were absent. The relevance of Ca2(+)-depleted virions for infection in vivo is discussed as well as the possibility that the Ca2(+)-binding sites may be parts of ion channels in the viral capsid. One possible RNA-binding site was found in the maps of both crystal types, and the same site could also be localized in the high-resolution map of native STNV.

Localization of DNA phosphorus in Tipula iridescent virus by oxygen plasma microincineration. II. Electron probe X-ray microanalysis of ash constituents.

Microincinerated, 1-micrometer sections of Tipula iridescent virus (TIV) crystals were analyzed quantitatively with a wave-length-dispersive electron microprobe to determine the chemical composition of high-resolution ash patterns previously obtained from ultrathin sections of the same specimen (Thomas RS: J Histochem Cytochem 29:379, 1981). Parallel analyses were performed on intact sections. In some cases, the same section and probe tracks were analyzed both before and after ashing. The principal elemental constituent of the ash was phosphorus, representing nearly all of the phosphorus found in the unashed sections. This confirmed the likely DNA origin of most of the ash. Other elements--sulfur, calcium, sodium, and perhaps carbon and nitrogen--found in relatively small concentrations in the ash, were partly due to either to incomplete ashing or to a particulate contaminant. Similar plasma ashing and analyses of sectioned model preparations of polymethacrylate containing dissolved triphenyl phosphate confirmed that phosphorus, by itself, could be retained as a stable ash. Analyses of polymethacrylate containing aluminum as well as phosphorus disclosed an unexpected artifact--aluminum inhibited the plasma ashing. These results suggested that the wavelength-dispersive probe, able to analyze for carbon and nitrogen as well as mineral elements, should be a generally useful tool in analyzing plasma microincineration phenomena, where macroscopic results do not apply. Relatively high beam intensity used throughout the probe analyses caused obvious damage to intact sections, particularly when they were mounted on thin-film supports. In contrast, the ash appeared quite stable. This suggested that plasma ashing of biological sections, converting them into mineralogical specimens, may be generally useful in probe analyses of mineral constituents, permitting greater sensitivity via higher beam currents, higher mineral concentrations, and lower backgrounds.

Physical characterization of PBCV virus by sedimentation field flow fractionation.

A virus (PBCV) from the symbiotic Chlorella-like algae of Paramecium bursaria has been characterized by sedimentation field flow fractionation (SdFFF). The method yields effective molecular weight M' (molecular weight X buoyancy factor) with a precision of better than 5% under a variety of experimental conditions. The value for M' is used to calculate true molecular weight values through combination with density values; densities were obtained from outside sources, from ultrasonic densitometry, and from sedimentation coefficients determined independently. The actual molecular weight is also determined through SdFFF analysis of the virus in carriers of different densities. Values for the particle diameter are obtained from SdFFF zone broadening studies. The various SdFFF characterization results are compared with molecular weight, particle size, and density values determined by conventional methods.

Dot-blot procedure with [32P]DNA probes for the sensitive detection of avocado sunblotch and other viroids in plants.

Avocado sunblotch viroid (ASBV) has been detected down to a level of about 20 pg per gram fresh weight of leaves by the use of a dot-blot hybridization procedure and partially purified nucleic acid extracts. Three [32P]DNA probes were compared, two prepared from full-length ASBV clones in the single-strand M13mp93 vector and the other by primer extension on purified ASBV. All three probes gave the same sensitivity of detection of ASBV. The methods developed have also been used successfully for the routine detection of potato spindle tuber viroid, citrus exocortis viroid, and coconut cadang cadang viroid.

In vitro 32P-labelling of viroid RNA for hybridization studies.

A method is described for the in vitro labelling of viroid RNA for use in hybridization studies. The citrus exocortis viroid (approximately 350 nucleotides) is degraded by hot formamide hydrolysis to fragments ranging from small oligonucleotides to near full lengths, and subsequently labelled to high specific activity by enzymatically attaching 32P to the 5'-end of each molecule. The cleavage step leaves 5' hydroxyl groups which allows the polynucleotide kinase to directly label the RNA fragments without prior enzymatic dephosphorylation. The method is simple, requires no special equipment, and provides a radioactive RNA probe sufficient for most types of hybridization studies.

Detection of double-stranded RNA by serologically specific electron microscopy.

Details of a procedure for detecting double-stranded RNA (dsRNA) in virus and viroid infected tissue extracts using serologically specific electron microscopy are given. A method for staining dsRNA, based on in situ formation of uranyl phosphate, that consistently permits the examination of dsRNA by electron microscopy without shadowing with heavy metals, is described. The method provides for routine assays for dsRNA in crude extracts without the variable results associated with shadowing procedures. DsRNA was found to accumulate in older leaves of sorghum systemically infected with sugarcane mosaic virus. In contrast, dsRNA was not detected in older cowpea leaves systemically infected with cowpea mosaic virus but was readily found in inoculated leaves 4 days post inoculation and young systemically infected leaves.

Electro-blot radioimmunoassay of virus-infected plant sap - a powerful new technique for detecting plant viruses.

A new technique for detecting viruses in plant sap is described. It consists of sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the infected plant sap, electrophoretic transfer of protein bands to activated paper by the Electro-Blot technique, the subsequent probing of the viral coat protein band by specific antiserum (prepared against intact virus), and detection of immune complex with 125 I-labelled protein A. The technique successfully detected tobacco mosaic virus at a sap dilution of 1 : 10,000, four strains of sugarcane mosaic virus (a potyvirus) in their perennial hosts infected for about 4 years, and five different isolated of potato leaf roll virus (a luteovirus). The latter virus occurs in extremely low concentration and is difficult to detect by the other known methods.