Time-resolved solution X-ray scattering of tobacco mosaic virus coat protein: kinetics and structure of intermediates

The kinetics of assembly and disassembly of tobacco mosaic virus coat protein (TMVP) following temperature jumps have been studied by small-angle X-ray scattering and turbidimetry. The structures of the principal aggregates of TMVP oligomers (A protein), intermediate size (helix I) and large size helical rods (helix II), have been characterized by their average radii of gyration of thickness, cross section, and shape obtained from the corresponding regimes of the small-angle scattering pattern. This structural information was obtained within seconds after the temperature-induced initiation of either polymerization or depolymerization and allowed us to detect transient intermediates. This methodology made it possible to observe and characterize the structure of a principal intermediate. Taken together with other kinetic information, these data suggest that polymerization of TMVP under virus self-assembly conditions may proceed via a single-layered helical nucleus that contains about 20 subunits. Previous studies have shown that overshoot polymerization of TMVP can occur and results in metastable long helical viruslike rods which subsequently depolymerize and then form short helical rods, depending on the conditions of the final equilibrium state. The longer rods (helix II) are overshoot polymers which form within seconds and contain 17 1/3 subunits per turn (helix IIB), in contrast to the subunit packing arrangement of 16 1/3 subunits per turn found in the shorter helical rods (helix IA). The latter packing arrangement is the one found in TMV. An overall polymerization scheme is proposed for the formation of these two helical forms of TMVP.     

Small angle X-ray scattering of dimeric yeast hexokinase in solution.

Small angle x-ray scattering measurements on dimeric yeast hexokinase B at pH 5.5 in acetate buffer yield a radius of gyration of 31.28 +/- 0.23 angstrom. This measured value is comparable to the radius of gyration of 31.5 angstrom calculated from the refined coordinates of the dimer in the BII crystal form. The hexokinase dimer found in the BI crystal form has a radius of gyration of 42 angstrom calculated from the atomic coordinates. Thus, the measured radius of gyration is consistent with the BII dimer being the predominant species in solution and rules out the existence of the BI dimer as a major species under these conditions.     

Differences of reconstitution process between tobacco mosaic virus and cucumber green mottle mosaic virus by synchrotron small angle X-ray scattering using low-temperature quenching

The differences of the reconstitution process of tobacco mosaic virus (TMV) and its mutant, cucumber green mottle mosaic virus (CGMMV) were investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. The reconstitution in an aqueous solution is completely stopped below 5°C. The TMV and CGMMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5°C on a series of solutions prepared by low-temperature quenching after incubation at 20°C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. The incubation of RNA and protein of CGMMV did not reconstitute at the initial reaction stages below 5 min and then began to reconstitute gradually. After 60 min, the radius of gyration for CGMMV reconstitution process reached almost the value for the initial stage of TMV reconstitution process. This is due to the fact the formation of double-layered disk in CGMMV protein is much slower than in TMV protein.     

Small angle neutron scattering studies of chromatin subunits in solution

Neutron scattering studies have been performed on dilute solutions of the fundamental subunit of chromatin, the nucleosome. The subunits contain approximately 195 base paris (bp) of DNA and histones H2A, H2B, H3, and H4. Measurements of the small angle scattering curves in various H2O/D2O solvents allow the contrast dependence of the radius of gyration of the subunits to be examined and give the mean scattering density of the particle. Further application of contrast variation to the higher angle scatter curves allows the contributions from the shape and internal structure of the subunits to be analyzed separately. From these results, we are able to propose a spherically averaged structure with most of the histones closely packed into a core of radius 3.2 nm surrounded by a loosely packed DNA-rich shell of 2.0 nm thickness resulting in a particle of 5.2 nm average radius. Model calculations for ellipsoids show that the outer shape of the subunit must have an axial ratio between 0.5 and 1.4 but is probably best described by more spherical particle. These results are correlated with the diffraction from chromatin films to provide an explanation for some of the diffraction rings.     

Small angle X-ray scattering reveals a compact intermediate in RNA folding

We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial 'electrostatic collapse' of the RNA is followed by slower rearrangements of elements that are initially mispositioned.     

Biophysical studies on the RNA cores of satellite tobacco mosaic virus

Satellite tobacco mosaic virus (STMV) was probed using a variety of proteases. Consequences of the degradation were analyzed using gel electrophoresis, quasi-elastic light scattering (QELS), and atomic force microscopy (AFM). Proteolysis rates of 30 minutes for complete degradation of the protein capsid, up to many hours, were investigated. With each protease, degradation of virions 17 nm in diameter was shown by QELS to result in particles of 10 nm diameter, which is that of the RNA core observed in the virion by x-ray diffraction analysis. This was verified by direct visualization with atomic force microscopy. Using QELS, it was further shown that freshly prepared RNA cores remain as individual, stable, 10-nm condensed particles for 12 to 24 h. Clusters of particles then formed, followed by very large aggregates of 500 to 1000 nm diameter. AFM showed that the aggregates were composed of groups of the condensed RNA cores and were not due to unfolding of the nucleic acid. No unfolding of the core particles into extended conformation was seen by AFM until the samples were heated well beyond 90 degrees C. Mass spectrometry of RNA core particles revealed the presence of a major polypeptide whose amino acid sequence corresponded to residues 2 through 25 of the coat protein. Amino acids 13 through 25 were previously observed to be in direct contact with the RNA and are presumably protected from protease digestion. Low resolution difference Fourier analyses indicated the courses of the remainders of the amino terminal strands (amino acids 2-12) in intact virions. Any individual strand appears to have several choices of path, which accounts for the observed disorder at high resolution. These positively charged strands, serving as virtual polyamines, engage the helical segments of RNA. The intimate association of amino acid residues 2 through 25 with RNA likely contributes to the stability of the condensed conformation of the nucleic acid cores.     

Replication of tobacco mosaic virus RNA

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

Biosynthesis of tobacco mosaic virus RNA in tobacco protoplasts

Changes in the number of protoplasts, viability, protein and chlorophyll contents and ribonucleases activity were studied in tobacco mesophyll protoplastsin vitro inoculated with tobacco mosaic virus (TMV). The number of protoplasts slowly increased during the cultivation period and the viability decreased from 95 to 67% in the control noninoculated protoplasts, and to 55% in the infected protoplasts. 30 h after inoculation the protein and chlorophyll contents strongly decreased to 25–30% and 17–19%, respectively, in comparison with contents 3 h after inoculation. The chlorophylla/b ratio decreased from 2.11 and 2.02 to 0.79 and 0.60 in healthy and infected protoplasts, respectively. The activities of ribonucleases in protoplasts quickly decreased during experiment but they were higher in infected than in noninfected protoplasts (between 20 to 30 h after inoculation they were 132 to 146% higher than that in healthy controls). These activities corresponded to the multiplication curve of TMV.     

Satellite tobacco mosaic virus RNA: structure and implications for assembly

The initial appearance of 45% of the single-stranded RNA of satellite tobacco mosaic virus in electron density maps suggested the entire RNA conformation could be delineated. Subsequent work has localized nearly 80% of the RNA as stem-loop elements. Connection of the stem-loops in the most efficient manner produces a persuasive model for the encapsidated RNA. This arrangement has significant implications for virus assembly and for the essential role of RNA.     

Structure of the RNA in tobacco mosaic virus

A model of the RNA of tobacco mosaic virus has been built using computer model-building techniques. The model has good stereochemistry, and fits the electron density map of the virus obtained by fiber diffraction methods considerably better than did earlier models. The three sugar rings in the asymmetric unit all have the A (3′-endo) conformation, One of the bases is in the syn conformation, a conformation observed only rarely in nucleic acid structures.     

Small angle X-ray scattering as a complementary tool for high-throughput structural studies

Structural crystallography and nuclear magnetic resonance (NMR) spectroscopy are the predominant techniques for understanding the biological world on a molecular level. Crystallography is constrained by the ability to form a crystal that diffracts well and NMR is constrained to smaller proteins. Although powerful techniques, they leave many soluble, purified structurally uncharacterized protein samples. Small angle X-ray scattering (SAXS) is a solution technique that provides data on the size and multiple conformations of a sample, and can be used to reconstruct a low-resolution molecular envelope of a macromolecule. In this study, SAXS has been used in a high-throughput manner on a subset of 28 proteins, where structural information is available from crystallographic and/or NMR techniques. These crystallographic and NMR structures were used to validate the accuracy of molecular envelopes reconstructed from SAXS data on a statistical level, to compare and highlight complementary structural information that SAXS provides, and to leverage biological information derived by crystallographers and spectroscopists from their structures. All the ab initio molecular envelopes calculated from the SAXS data agree well with the available structural information. SAXS is a powerful albeit low-resolution technique that can provide additional structural information in a high-throughput and complementary manner to improve the functional interpretation of high-resolution structures.     

Solution X-ray scattering study of reconstitution process of tobacco mosaic virus particle using low-temperature quenching

The reconstitution process of tobacco mosaic virus (TMV) was investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. TMV assembly in an aqueous solution is completely stopped below 5 degrees C. The TMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5 degrees C on a series of solutions prepared by low-temperature quenching after incubation either at 15, 20 or 25 degrees C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. In order to account the time course of SAXS profiles in terms of the elongation of TMV assembly, a model calculation was performed to simulate the Guinier plot, the Kratky plot and the distance distribution function by applying Glatter's multibody method using models that were constituted of the spheres representing a column of piled two-layer disks of TMV-protein. The three simulated functions thus obtained support the conclusion derived from the three functions calculated from the experimental results that the incubation of the RNA and protein of TMV began to reconstitute TMV instantly after mixing, proceeded steeply to a long rod, and then extended asymptotic to the full length of the TMV particle. This process is in good agreement with that obtained from electron microscopic studies.     

Expression of tobacco mosaic virus RNA in transgenic plants

Summary Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in planta.