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A method of Transitional Automorphic Mapping of the Genome on Itself (TAMGI) is aimed at combining detection and reconstruction of correlational and quasi-periodic motifs in the viral genomic RNA/DNA sequences. The motifs reconstructed by TAMGI are robust with respect to indels and point mutations and can be tried as putative therapeutic targets. We developed and tested the relevant theory and statistical criteria for TAMGI applications. The applications of TAMGI are illustrated by the study of motifs in the genomes of the severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2 (the latter coronavirus SARS-CoV-2 being responsible for the COVID-19 pandemic) packaged within filament-like helical capsid. Such ribonucleocapsid is transported into spherical membrane envelope with incorporated spike glycoproteins. Two other examples concern the genomes of viruses with icosahedral capsids, satellite tobacco mosaic virus (STMV) and bacteriophage PHIX174. A part of the quasi-periodic motifs in these viral genomes was evolved due to weakly specific cooperative interaction between genomic ssRNA/ssDNA and nucleocapsid proteins. The symmetry of the capsids leads to the natural selection of specific quasi-periodic motifs in the related genomic sequences. Generally, TAMGI provides a convenient tool for the study of numerous molecular mechanisms with participation of both quasi-periodic motifs and complete repeats, the genome organization, contextual analysis of cis/trans regulatory elements, data mining, and correlations in the genomic sequences., 22 pages, 4 figures

It was found that the crystals of at least a dozen different proteins could be thoroughly stained to an intense color with a panel of dyes. Many, if not most, of the stained protein crystals retained the dyes almost indefinitely when placed in large volumes of dye-free mother liquor. Dialysis experiments showed that most of the dyes that were retained in crystals also bound to the protein when free in solution; less frequently, some dyes bound only in the crystal. The experiments indicated a strong association of the dyes with the proteins. Four protein crystals were investigated by X-ray diffraction to ascertain the mode of binding. These were crystals of lysozyme, thaumatin, trypsin inhibited with benzamidine and satellite tobacco mosaic virus. In 30 X-ray analyses of protein crystal–dye complexes, in only three difference Fourier maps was any difference electron density present that was consistent with the binding of dye molecules, and even in these three cases (thaumatin plus thioflavin T, xylene cyanol and m-cresol purple) the amount of dye observed was inadequate to explain the intense color of the crystals. It was concluded that the dye molecules, which are clearly inside the crystals, are disordered but are paradoxically tightly bound to the protein. It is speculated that the dyes, which exhibit large hydrophobic cores and peripheral charged groups, may interact with the crystalline proteins in the manner of conventional detergents.

The RNA genomes of viruses likely undergo multiple functionally important conformational changes during their replication cycles, changes that are poorly understood at present. We used two complementary in-solution RNA structure probing strategies (SHAPE-MaP and RING-MaP) to examine the structure of the RNA genome of satellite tobacco mosaic virus inside authentic virions and in a capsid-free state. Both RNA states feature similar three-domain architectures in which each major replicative function-translation, capsid coding, and genome synthesis-fall into distinct domains. There are, however, large conformational differences between the in-virion and capsid-free states, primarily in one arm of the central T domain. These data support a model in which the packaged capsid-bound RNA is constrained in a local high-energy conformation by the native capsid shell. The removal of the viral capsid then allows the RNA genome to relax into a more thermodynamically stable conformation. The RNA architecture of the central T domain thus likely changes during capsid assembly and disassembly and may play a role in genome packaging.

Three-dimensional RNA domain reconstruction is important for the assembly, disassembly and delivery functionalities of a packed proteinaceus capsid. However, to date, the self-association of RNA molecules is still an open problem. Recent chemical probing reports provide, with high reliability, the secondary structure of diverse RNA ensembles, such as those of viral genomes. Here, we present a method for reconstructing the complete 3D structure of RNA genomes, which combines a coarse-grained model with a subdomain composition scheme to obtain the entire genome inside proteinaceus capsids based on secondary structures from experimental techniques. Despite the amount of sampling involved in the folded and also unfolded RNA molecules, advanced microscope techniques can provide points of anchoring, which enhance our model to include interactions between capsid pentamers and RNA subdomains. To test our method, we tackle the satellite tobacco mosaic virus (STMV) genome, which has been widely studied by both experimental and computational communities. We provide not only a methodology to structurally analyze the tertiary conformations of the RNA genome inside capsids, but a flexible platform that allows the easy implementation of features/descriptors coming from both theoretical and experimental approaches.

The assembly and maturation of viruses with icosahedral capsids must be coordinated with icosahedral symmetry. The icosahedral symmetry imposes also the restrictions on the cooperative specific interactions between genomic RNA/DNA and coat proteins that should be reflected in quasi-regular segmentation of viral genomic sequences. Combining discrete direct and double Fourier transforms, we studied the quasi-regular large-scale segmentation in genomic sequences of different ssRNA, ssDNA, and dsDNA viruses. The particular representatives included satellite tobacco mosaic virus and the strains of satellite tobacco necrosis virus, STNV-C, STNV-1, STNV-2, Escherichia phages MS2, phiX174, alpha3, and HK97, and Simian virus 40. In all their genomes, we found the significant quasi-regular segmentation of genomic sequences related to the virion assembly and the genome packaging within icosahedral capsid. We also found good correspondence between our results and available cryo-electron microscopy data on capsid structures and genome packaging in these viruses. Fourier analysis of genomic sequences provides the additional insight into mechanisms of hierarchical genome packaging and may be used for verification of the concepts of 3-fold or 5-fold intermediates in virion assembly. The results of sequence analysis should be taken into account at the choice of models and data interpretation. They also may be helpful for the development of antiviral drugs., Comment: 55 pages, 14 figures

Viral RNA structure prediction is a valuable tool for development of drugs against viral disease. This work discusses different approaches to predicting encapsidated viral RNA and highlights satellite tobacco mosaic virus (STMV) RNA as a model system with excellent crystallography data. Fundamentally important issues for debate include thermodynamic versus kinetic control of virus assembly and the possible consequences of quasi-species in the primary structure on RNA secondary structure prediction of a single structure or an ensemble of structures. Multiple computational tools and chemical reagents are now available for improved viral RNA structure prediction. Two different predicted structures for encapsidated STMV RNA result from differences in three main areas: a different approach and philosophy to studying encapsidated viral RNA, an emphasis on different RNA motifs, and technical differences in computational methods and chemical reagents. The experiments with traditional chemical probing and SHAPE reagents are compared in terms of chemistry, results, and interpretation for STMV RNA as well as other RNA protein assemblies, such as the 5'UTR of HIV and the ribosome. This discussion of the challenges of viral RNA structure prediction will lead to new experiments and improved future predictions for viral RNA.

Satellite tobacco mosaic virus(STMV) is among the smallest viruses, having 60 identical subunits arranged withT= 1 icosahedral symmetry. Its crystal structure was solved at 290 K and was refined using, in part, crystals grown in microgravity. Electron-density maps revealed nearly 57% of the genomic ssRNA. Using six flash-cooled crystals, diffraction data were recorded to 1.4 Å resolution and independent refinements of the STMV model were carried outversusthe previous 1.8 Å resolution data representing merged data from 21 crystals (271 689 unique reflections), data consisting of corresponding reflections to 1.8 Å resolution from the cooled crystals and 1.4 Å resolution data from the cooled crystals comprised of 570 721 unique reflections. Models were independently refined with full NCS constraints using the programCNSand in restrained mode using the programsCNS,REFMAC5 andSHELX-97, with the latter two procedures including anisotropic temperature factors. Significant additional structural detail emerged from the analyses, including a unique cation and anion arrangement on fivefold axes and a precise assessment of icosahedral symmetry exactness in the crystal lattice. STMV represents the highest resolution native virus structure currently known by a substantial margin, and it permits the evaluation of a precise atomic model of a spherical virus at near-atomic resolution for the first time.

Satellite viruses (SVs) are subviral pathogens that are entirely dependent upon the replication machinery of their helper viruses. There are only four known plant SVs: satellite tobacco necrosis virus, satellite tobacco mosaic virus, satellite panicum mosaic virus, and satellite maize white line mosaic virus. These SVs have positive-sense single-stranded RNA genomes of 800–1400 nt that are encapsidated within ~17-nm T =1 icosahedral virions. SVs, in contrast to satellite-RNA or -DNA, encode a capsid protein for independent genome packaging of the cognate SV RNA. The unusually small and compact nature of these subviral agents has resulted in their use as models for fundamental virology studies, including gene expression, genome packaging, and virion structure.

Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3′ terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging.

The complete structure of the genomic material inside a virus capsid remains elusive, although a limited amount of symmetric nucleic acid can be resolved in the crystal structure of 17 icosahedral viruses. The negatively charged sugar-phosphate backbone of RNA and DNA as well as the large positive charge of the interior surface of the virus capsids suggest that electrostatic complementarity is an important factor in the packaging of the genomes in these viruses. To test how much packing information is encoded by the electrostatic and steric envelope of the capsid interior, we performed extensive all-atom molecular dynamics (MD) simulations of virus capsids with explicit water molecules and solvent ions. The model systems were two small plant viruses in which significant amounts of RNA has been observed by X-ray crystallography: satellite tobacco mosaic virus (STMV, 62% RNA visible) and satellite tobacco necrosis virus (STNV, 34% RNA visible). Simulations of half-capsids of these viruses with no RNA present revealed that the binding sites of RNA correlated well with regions populated by chloride ions, suggesting that it is possible to screen for the binding sites of nucleic acids by determining the equilibrium distribution of negative ions. By including the crystallographically resolved RNA in addition to ions, we predicted the localization of the unresolved RNA in the viruses. Both viruses showed a hot-spot for RNA binding at the 5-fold symmetry axis. The MD simulations were compared to predictions of the chloride density based on nonlinear Poisson-Boltzmann equation (PBE) calculations with mobile ions. Although the predictions are superficially similar, the PBE calculations overestimate the ion concentration close to the capsid surface and underestimate it far away, mainly because protein dynamics is not taken into account. Density maps from chloride screening can be used to aid in building atomic models of packaged virus genomes. Knowledge of the principles of genome packaging might be exploited for both antiviral therapy and technological applications.

Agarose gel electrophoresis, circular dichroism and differential scanning calorimetry showed that single-stranded RNA from satellite tobacco mosaic virus transforms from a conformationally ‘closed state’ at 4 � C to a more conformationally ‘open state’ at 65 � C. The transition is reversible and shows no hysteresis. Atomic force microscopy (AFM) allowed visualization of the two states and indicated that the conformationally ‘closed state’ probably corresponds to the native encapsidated conformation, and that the ‘open state’ represents a conformation, characterized as short, thick chains of domains, as a consequence of the loss of tertiary interactions. Heating from 75 � Ct o 85 � C in the presence of EDTA was necessary to further unravel the ‘open’ conformation RNA into extended chains of lengths >280nm. Virus exposed to low concentrations of phenol at 65 � C, extruded RNA as distinctive ‘pigtails’ in a synchronous fashion, and these ‘pigtails’ then elongated, as the RNA was further discharged by the particles. Moderate concentrations of phenol at 65 � C produced complete disruption of virions and only remains of decomposed particles and disordered RNA were evident. AFM images of RNA emerging from disrupted virions appear most consistent with linear arrangements of structural domains.

Coarse-grained elastic network models elucidate the fluctuation dynamics of proteins around their native conformations. Low-frequency collective motions derived by simplified normal mode analysis are usually involved in biological function, and these motions often possess noteworthy symmetries related to the overall shape of the molecule. Here, insights into these motions and their frequencies are sought by considering continuum models with appropriate symmetry and boundary conditions to approximately represent the true atomistic molecular structure. We solve the elastic wave equations analytically for the case of spherical symmetry, yielding a symmetry-based classification of molecular motions together with explicit predictions for their vibrational frequencies. We address the case of icosahedral symmetry as a perturbation to the spherical case. Applications to lumazine synthase, satellite tobacco mosaic virus, and brome mosaic virus show that the spherical elastic model efficiently provides insights on collective motions that are otherwise obtained by detailed elastic network models. A major utility of the continuum models is the possibility of estimating macroscopic material properties such as the Young's modulus or Poisson's ratio for different types of viruses.

Considered is the coarse-grained modeling of icosahedral viruses in terms of a three-dimensional lattice (the digital modeling lattice) selected among the projected points in space of a six-dimensional icosahedral lattice. Backbone atomic positions (Cα's for the residues of the capsid and phosphorus atoms P for the genome nucleotides) are then indexed by their nearest lattice point. This leads to a fine-grained lattice point characterization of the full viral chains in the backbone approximation (denoted as digital modeling). Coarse-grained models then follow by a proper selection of the indexed backbone positions, where for each chain one can choose the desired coarseness. This approach is applied to three viruses, the Satellite tobacco mosaic virus, the bacteriophage MS2 and the Pariacoto virus, on the basis of structural data from the Brookhaven Protein Data Bank. In each case the various stages of the procedure are illustrated for a given coarse-grained model and the corresponding indexed positions are listed. Alternative coarse-grained models have been derived and compared. Comments on related results and approaches, found among the very large set of publications in this field, conclude this article.

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.

Three new crystal forms of satellite panicum mosaic virus (SPMV) were grown and their structures solved from X-ray diffraction data using molecular replacement techniques. The crystals were grown under conditions of pH and ionic strength that were appreciably different then those used for the original structure determination. In rhombohedral crystals grown at pH 8.5 and low ionic strength PEG 3350 solutions, Fourier syntheses revealed segments, ten amino acid residues long, of amino-terminal polypeptides not previously seen, as well as masses of electron density within concavities on the interior of the capsid, which appeared in the neighborhoods of icosahedral five- and threefold axes. The densities were compatible with secondary structural domains of RNA, and they included a segment of double helical RNA of about four to five base pairs oriented, at least approximately, along the fivefold axes. The distribution of RNA observed for SPMV appears to be distinctly different than the encapsidated nucleic acid conformation previously suggested for another satellite virus, satellite tobacco mosaic virus. This study further shows that analysis of viruses in crystals grown under different chemical conditions may reveal additional information regarding the structure of encapsidated RNA. © 2006 Elsevier Inc. All rights reserved.

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26°C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins. © 2005 Elsevier Ltd. All rights reserved.

We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well-characterized bridging species for the Solanaceae plant family.

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle χ2,1 for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

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.

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.

Isolates of tobacco mild green mosaic tobamovirus (TMGMV) were obtained from 58 plants of Nicotiana glauca in southern California and placed in one of two groups (Small type and Large type) based on the size of the subgenomic RNA for the coat protein (CP). The CP sequence differed by no more than one amino acid for the two types, and the Small type was identical to that published for TMGMV. Thirty-six of the isolates had a double-stranded (ds)RNA profile that matched that of type TMGMV, and the nucleotide sequence of the 3' untranslated region (3'UTR) of six of these isolates was similar to the published sequence of TMGMV. Twenty-two isolates had a larger dsRNA for the CP subgenomic RNA. Six of these were sequenced and all had a repeat sequence of between 147 and 165 bases in the part of the 3'UTR that is involved in the formation of pseudoknots. These novel but common isolates are predicted to have six rather than three pseudoknots. Small types (three pseudoknots=type TMGMV) yielded twice as much virus after purification as Large types (six pseudoknots). The two groups of isolates could be distinguished in N. rustica (Large type, but not Small type gave a systemic infection), and N. clevelandii (Small type but not Large type induced systemic lethal necrosis). Almost all isolates of TMGMV used in this study were initially associated with satellite tobacco mosaic virus (STMV), and both types supported STMV experimentally.

Atomic force microscopy (AFM) has been used to visualize events arising from the formation of intervening metastable phases at the surfaces of macromolecular crystals growing from solution. Crystals investigated were of the proteins canavalin, thaumatin, lipase, xylanase, and catalase, crystals of transfer RNA, and crystals of satellite tobacco mosaic virus. The appearance of aggregates on crystal surfaces was observed. The aggregates we infer to originate from liquid-protein droplets. These were particularly evident in freshly mixed mother liquor solutions. Droplets, upon sedimentation, have two possible fates. In some cases they immediately restructured as crystalline, multilayer stacks whose development was guided by, and contiguous with the underlying lattice. These contributed to the ordered growth of the crystal by serving as sources of growth steps. In other cases, liquid-protein droplets formed distinct microcrystals, somehow discontinuous with the underlying lattice, and these were subsequently incorporated into the growing substrate crystal. Scarring experiments with the AFM tip indicated that, detached from the crystal, molecules do not dissolve in the fluid phase but form metastable liquid-protein droplets with a potential to rapidly crystallize on the crystal surface. The molecular structure of the growth steps for thaumatin and lipase protein crystals were deduced. There is no step roughness due to thermal fluctuations, and each protein molecule which incorporated into the step edge remained. Growth steps propagate by addition of individual molecules which form subkinks of different size on the step edge.

Atomic force microscopy (AFM) has been used to visualize events arising from the formation of intervening metastable phases at the surfaces of macromolecular crystals growing from solution. Crystals investigated were of the proteins canavalin, thaumatin, lipase, xylanase, and catalase, crystals of transfer RNA, and crystals of satellite tobacco mosaic virus. Two types of aggregates were observed. The first were small, linear and branched aggregates, perhaps fractile in structure. These were incorporated into growing crystals as impurities, and they produced defects of various kinds. The second aggregate form we infer to be liquid-protein droplets which were particularly evident in freshly mixed protein-precipitant solutions. Droplets, upon sedimentation, have two possible fates. In some cases they immediately restructured as crystalline multilayer stacks whose development was guided by and contiguous with the underlying lattice. These contributed to the ordered growth of the crystal by serving as sources of growth steps. In other cases, liquid-protein droplets formed distinct microcrystals, somehow discontinuous with the underlying lattice, and these were subsequently incorporated into the growing substrate crystal with the formation of defects. Scarring experiments with the AFM tip indicated that liquid-protein droplets with the potential to rapidly crystallize were a consequence of concentration instabilities near the crystal's surfaces. The AFM study suggests that phase separation and the appearance of aggregates having limited order is a common occurrence in supersaturated macromolecular solutions such as the protein-precipitant solutions used for crystallization.

Mathews, D. M., and Dodds, J. A. 1998. Naturally occurring variants of satellite tobacco mosaic virus. Phytopathology 88:514-519. Four natural variants of sate llite tobacco mosaic virus (STMV) were compared with each other and with the type strain. Differences were detected in double-stranded RNA, single-stranded RNA, and virion electrophoretic mobility patterns, while the size and antigenicity of the coat protein were similar for all. RNase protection assays detected differences in the genomes of each of the four new variants, which differed not only from each other, but also from that of type STMV. Infectious RNA transcripts were made from complementary DNA clones of one variant (STMV 10) with a genome apparently smaller than that of type STMV. A 71-base deletion in the region that contains the 6.8-kDa protein in type STMV was detected by sequence analysis of the STMV 10 clones, a result that is confirmed by the lack of a 6.8-kDa in vitro translation product for STMV 10. Only minor sequence differences exist elsewhere in the genome compared with that of type STMV. Type STMV and STMV 10 each successfully cross-protected against the other when tobacco plants were inoculated 10 days apart. Satellite tobacco mosaic virus (STMV) was discovered in 1986 in plants of Nicotiana glauca Graham (tree tobacco) that were also infected with tobacco mild green mosaic virus (TMGMV, also known as TMV-U5) (21,22). STMV is a 17-nm, spherical virus with T = 1 symmetry (6) and is dependent upon tobamoviruses for its replication. It produces its own 17.5-kDa coat protein from a plus-sense, single-stranded (ss) RNA genome of 1,059 nucleotides (14). An overlapping open reading frame (ORF) for a 6.8-kDa protein of unknown function is present upstream of the coat protein gene in the genome of the type strain (14). The first isolate of STMV to be cloned and sequenced was designated the “type” strain (14), but it has become apparent through genome comparisons in which RNase protection assays (RPAs) were used that STMV isolates are genetically quite diverse (8,9) and that a dominant “strain” may not exist in nature. However, few of these isolates were fully sequenced or characterized for other viral properties so that a broad comparison could be performed. The present study was initiated to better understand variability among isolates of STMV and to evaluate whether the type strain is representative of the virus. Four field isolates of STMV among a collection of isolates from individual plants of N. glauca infected with TMGMV in Riverside County, CA, were identified by double-stranded (ds) RNA electrophoretic mobilities as different from type STMV (three had larger dsRNA, and one had smaller). They were originally isolated by R. Valverde, who provided them for use in this study. The isolates, designated STMV 6, 8, 10, and 19 (isolate numbers are not the same as those used by others, including Kurath et al. [8,9] and Valverde and Dodds [22]), were studied for general viral characters, and one was chosen for cloning and sequencing. Analysis of these isolates has provided additional information on the natural diversity of STMV, including evidence for the dispensability of the 6.8-kDa protein.

The genome of satellite tobacco mosaic virus (STMV) adapted to tobacco mosaic virus (TMV), tomato mosaic virus or green tomato atypical mosaic virus consistently had two single base deletions at positions 1 and 61, corresponding to bases A and G, respectively, as compared to the type-strain genome which is naturally adapted to tobacco mild green mosaic virus (TMGMV). Transcript RNAs (STMV(TMV)) from clone pSTMV(TMV) which captured the deletions at positions 1 and 61 were infectious when co-inoculated to tobacco plants with either TMV or TMGMV at infection frequencies of > 90%. Two new STMV variants were created to investigate whether both deletions were essential for adaptation to TMV. These were STMV(TMGMV) deltaA1, which had the A at position 1 (A1) deleted, and STMV(TMGMV) deltaG61, which lacked G61. STMV(TMGMV) deltaA1 was infectious (75% frequency) in the presence of either TMV or TMGMV. Virion RNA of STMV(TMGMV) deltaA1 lost G61 after one infection cycle with TMV. This deletion did not occur in co-infections with TMGMV. STMV(TMGMV) deltaG61, like the clone STMV(TMGMV), was infectious (100% frequency) with TMGMV but TMV did not support this clone. When Nicotiana benthamiana protoplasts were transfected with STMV(TMGMV), STMV(TMGMV) deltaA1 or STMV(TMV), STMV replicated when TMGMV was the helper virus. STMV(TMV) and STMV(TMGMV) deltaA1 replicated in the presence of helper TMV, but STMV(TMGMV) did not, the same result as in whole plants. The deletion of A1 is thus essential for initial STMV adaptation to TMV and the eventual deletion of G61 is a predicted additional change.

The molecular structure of satellite tobacco mosaic virus (STMV) has been refined to 1.8 A resolution using X-ray diffraction data collected from crystals grown in microgravity. The final R value was 0.179 and R free was 0.184 for 219,086 independent reflections. The final model of the asymmetric unit contained amino acid residues 13 to 159 of a coat protein monomer, 21 nucleotides, a sulfate ion, and 168 water molecules. The nucleotides were visualized as 30 helical segments of nine base-pairs with an additional base stacked at each 3′ end, plus a “free” nucleotide, not belonging to the helical segments, but firmly bound by the protein. Sulfate ions are located exactly on 5-fold axes and each is coordinated by ten asparagine side-chains. Of the 10,080 structural waters, 168 per asymmetric unit, about 20% serve to bridge the macromolecular components at protein-protein and protein-nucleic acid interfaces. Binding of RNA to the protein involves some salt linkages, particularly to the phosphate of the free nucleotide, but the major contribution is from an intricate network of hydrogen bonds. There are numerous water molecules in the RNA-protein interface, many serving as intermediate hydrogen bond bridges. The sugar-phosphate backbone contributes most of the donors and acceptors for the RNA. The helical RNA conformation is nearest that of A form DNA. The central region of a helical segment is most extensively involved in contacts with protein, and exhibits low thermal parameters which increase dramatically toward the ends. The visible RNA represents approximately 59% of the total nucleic acid in the virion and is derived from the single-stranded genome, which has folded upon itself to form helical segments. Linking of the helices and the free nucleotides in a contiguous and efficient manner severely restricts the disposition of the remaining, unseen nucleic acid. Using the remaining nucleotides it is possible to fold the RNA according to motifs that provide a periodic distribution of RNA structural elements compatible with the icosahedrally symmetrical arrangement seen in the crystallographic structure. The intimate relationship between protein and nucleic acid in STMV suggests an assembly pathway based on the cooperative and coordinated co-condensation of RNA with capsid protein dimers.

The evolution of surface morphology and step dynamics during growth of rhombohedral crystals of the protein canavalin and crystals of the cubic satellite tobacco mosaic virus (STMV) have been investigated for the first time by in situ atomic force microscopy. These two crystals were observed to grow by very different mechanisms. Growth of canavalin occurs on complex vicinal hillocks formed by multiple, independently acting screw dislocations. Small clusters were observed on the terraces. STMV on the other hand, was observed to grow by 2D nucleation of islands. No dislocations were found on the crystal. The results are used to determine the growth mechanisms and estimate the fundamental materials parameters. The images also illustrate the important mechanism of defect incorporation and provide insight to the processes that limit the growth rate and uniformity of these crystals.

In the course of time-lapse video and atomic force microscopy (AFM) investigations of macromolecular crystal growth, we frequently observed the sedimentation of microcrystals and three-dimensional nuclei onto the surfaces of much larger, growing protein or virus crystals. This was followed by the direct incorporation over time of the smaller crystals into the bulk of the larger crystals. In some cases, clear indications were present that upon absorption of the small crystal onto the surface of the larger, there was proper alignment of the respective lattices, and consolidation proceeded without observable defect formation, i.e., the two lattices knitted together without discontinuity. In the case of at least one virus crystal, cubic satellite tobacco mosaic virus (STMV), addition of three-dimensional nuclei and subsequent expansion provided the principal growth mechanism at high supersaturation. This process has not been reported for growth from solution of conventional crystals. In numerous other instances, the lattices of the small and larger crystals were obviously misaligned, and incorporation occurred with the formation of some defect. This phenomenon of small crystals physically embedded in larger crystals could only degrade the overall diffraction and materials properties of macromolecular crystals.

We have developed a model for the secondary structure of the 1058-nucleotide plus-strand RNA genome of the icosahedral satellite tobacco mosaic virus (STMV) using nucleotide-resolution SHAPE chemical probing of the viral RNA isolated from virions and within the virion, perturbation of interactions distant in the primary sequence, and atomic force microscopy. These data are consistent with long-range base pairing interactions and a three-domain genome architecture. The compact domains of the STMV RNA have dimensions of 10 to 45 nm. Each of the three domains corresponds to a specific functional component of the virus: The central domain corresponds to the coding sequence of the single (capsid) protein encoded by the virus, whereas the 5′ and 3′ untranslated domains span signals essential for translation and replication, respectively. This three-domain architecture is compatible with interactions between the capsid protein and short RNA helices previously visualized by crystallography. STMV is among the simplest of the icosahedral viruses but, nonetheless, has an RNA genome with a complex higher-order structure that likely reflects high information content and an evolutionary relationship between RNA domain structure and essential replicative functions.

Some small icosahedral RNA viruses (e.g., MS2) require a specific packaging signal for the formation of virus particles. Others (e.g., cowpea chlorotic mottle virus (CCMV); Pariacoto virus (PaV)) are able to encapsidate a wide variety of RNAs, forming virus-like particles (VLPs) whose structure are only slightly different from that of the wild-type virus. We have determined the structure of the genomic RNA of satellite tobacco mosaic virus (STMV) in an in vitro transcript (see image below). This structure is very different from the structure of STMV RNA probed inside the virus by Schroeder et al. (BJ 101:167 (2011)), which consists of a string of stem-loops connected by single-stranded regions. We have also developed all-atom models for three viruses: PaV (using a hypothetical secondary structure and a non-viral sequence); STMV (using the true sequence and the Schroeder secondary structure); and MS2 (using the true sequence and a hypothetical secondary structure). In this talk, we discuss the implications of these secondary structures and three-dimensional models for the pathways of viral assembly.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Detailed structures are now available for three plant satellite viruses, satellite tobacco necrosis virus (STNV), satellite tobacco mosaic virus (STMV), and satellite panicum mosaic virus (SPMV). It is, therefore, possible to compare the tertiary structure of viral protein subunits, their quaternary interactions, and the interactions of protein subunits with the RNA genome. This analysis indicates that, in spite of common function and preservation of a 'jelly-roll' motif in the protein monomer, the three viruses are remarkably different. The differences include the arrangement of secondary structural elements, Interactions of adjacent subunits, and the disposition of subunits relative to icosahedral symmetry axes. In each of the three viruses, however, the narrow end of the jelly roll forms fivefold contacts. The fivefold protein interactions are organized about a Ca2+ ion for STNV, an anion for STMV, and, apparently, neither of these for SPMV. Low-resolution neutron diffraction studies using H2O/D2O solvent contrast variation revealed the general location of the RNA genome within the STNV. In the case of SPMV, regions of electron density on the interior of the capsid could be assigned to RNA, although it was not possible to model the nucleic acid. Only for STMV was nucleic acid visible in ejection density maps, and this was manifested as double-helical RNA segments associated with each coat protein dimer. The observations presented here provide no support for any common evolutionary relationship. © 1995 Academic Press, Inc.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.

Satellite tobacco mosaic virus (STMV) is an icosahedral T=1 single-stranded RNA virus with a genome containing 1058 nucleotides. X-ray crystallography revealed a structure containing 30 double-helical RNA segments, with each helix having nine base pairs and an unpaired nucleotide at the 3' end of each strand. Based on this structure, Larson and McPherson proposed a model of 30 hairpin-loop elements occupying the edges of the icosahedron and connected by single-stranded regions. More recently, Schroeder et al. have combined the results of chemical probing with a novel helix searching algorithm to propose a specific secondary structure for the STMV genome, compatible with the Larson-McPherson model. Here we report an all-atom model of STMV, using the complete protein and RNA sequences and the Schroeder RNA secondary structure. As far as we know, this is the first all-atom model for the complete structure of any virus (100% of the atoms) using the natural genomic sequence. © 2012 Elsevier Inc.

Knowledge about structure and dynamics of the important biological macromolecules — proteins, nucleic acids, lipids and sugars — helps to understand their function. Atomic-resolution structures of macromolecules are routinely captured with X-ray crystallography and other techniques. In this thesis, simulations are used to explore the dynamics of the molecules beyond the static structures. Viruses are machines constructed from macromolecules. Crystal structures of them reveal little to no information about their genomes. In simulations of empty capsids, we observed a correlation between the spatial distribution of chloride ions in the solution and the position of RNA in crystals of satellite tobacco necrosis virus (STNV) and satellite tobacco mosaic virus (STMV). In this manner, structural features of the non-symmetric RNA could also be inferred. The capsid of STNV binds calcium ions on the icosahedral symmetry axes. The release of these ions controls the activation of the virus particle upon infection. Our simulations reproduced the swelling of the capsid upon removal of the ions and we quantified the water permeability of the capsid. The structure and dynamics of the expanded capsid suggest that the disassembly is initiated at the 3-fold symmetry axis. Several experimental methods require biomolecular samples to be injected into vacuum, such as mass-spectrometry and diffractive imaging of single particles. It is therefore important to understand how proteins and molecule-complexes respond to being aerosolized. In simulations we mimicked the dehydration process upon going from solution into the gas phase. We find that two important factors for structural stability of proteins are the temperature and the level of residual hydration. The simulations support experimental claims that membrane proteins can be protected by a lipid micelle and that a non-membrane protein could be stabilized in a reverse micelle in the gas phase. A water-layer around virus particles would impede the signal in diffractive experiments, but our calculations estimate that it should be possible to determine the orientation of the particle in individual images, which is a prerequisite for three-dimensional reconstruction. BMC B41, 25/5, 9:15

The secondary structure of satellite tobacco mosaic virus (STMV) RNA was predicted using computer simulations of RNA folding. The analogies of structural elements in the 3' end untranslated regions (3'-UTR) of tobamoviral RNAs were analysed. In addition to the tRNA-like structure and pseudoknot stalk, which are found in all known RNAs of tobamoviruses and STMV, another region of stable consecutive pseudoknots was predicted in the 3'-UTR of STMV RNA. A similar pattern of repeated structural units, containing pseudoknot stalks and parts of the tRNA-like structure, was also found in odontoglossum ringspot virus (ORSV) RNA 3'-UTR. The predictions on the structure are supported by sequence comparisons which point to an important functional role of 3' terminal pseudoknots in STMV RNA as well as in other tobamoviral RNAs. The possible participation of pseudoknotted structures in the interactions with coat protein in STMV is discussed.

A multiscale mathematical and computational approach is developed that captures the hierarchical organization of a microbe. It is found that a natural perspective for understanding a microbe is in terms of a hierarchy of variables at various levels of resolution. This hierarchy starts with the N -atom description and terminates with order parameters characterizing a whole microbe. This conceptual framework is used to guide the analysis of the Liouville equation for the probability density of the positions and momenta of the N atoms constituting the microbe and its environment. Using multiscale mathematical techniques, we derive equations for the co-evolution of the order parameters and the probability density of the N-atom state. This approach yields a rigorous way to transfer information between variables on different space-time scales. It elucidates the interplay between equilibrium and far-from-equilibrium processes underlying microbial behavior. It also provides framework for using coarse-grained nanocharacterization data to guide microbial simulation. It enables a methodical search for free-energy minimizing structures, many of which are typically supported by the set of macromolecules and membranes constituting a given microbe. This suite of capabilities provides a natural framework for arriving at a fundamental understanding of microbial behavior, the analysis of nanocharacterization data, and the computer-aided design of nanostructures for biotechnical and medical purposes. Selected features of the methodology are demonstrated using our multiscale bionanosystem simulator DeductiveMultiscaleSimulator. Systems used to demonstrate the approach are structural transitions in the cowpea chlorotic mosaic virus, RNA of satellite tobacco mosaic virus, virus-like particles related to human papillomavirus, and iron-binding protein lactoferrin.

Four icosahedral RNA viruses are considered: the cowpea chlorotic mottle virus, the satellite tobacco mosaic virus, the pariacoto virus and the MS2 bacteriophage. The validity of the phenomenological rules derived in previous publications (crystallographic scaling, indexed forms enclosing axial-symmetric clusters, packing lattices of viral crystals) is confirmed and shown to apply equally well to the coat proteins as to the (ordered) RNA chains.

Order Parameters (OPs) characterizing the structure and organization of bio-nanosystems (BNSs) are presented. Deductive all-atom multiscale techniques imply that structural transitions in several BNSs can be simulated via the slow, temporal OP dynamics, which co-evolves with a quasi-equilibrium probability density for rapidly fluctuating atomic configurations. This yields a force-field based algorithm that allows for all-atom BNS simulations with high CPU efficiency. Salient features of a set of OPs including their construction, emergence and dynamical completeness are discussed. These are critical in probing the free-energy landscapes underlying structural transitions. The computational algorithm is implemented via a software, denoted SimNanoWorld, that we demonstrate in applications to macromolecules and macromolecular assemblies. This includes simulating the dynamics of viral RNA and RNA-protein complexes in Satellite Tobacco Mosaic Virus (STMV) over a range of electrolytes (1:1 and 2:1) and temperatures (300K-600K). Another example demonstrates the application of OPs in probing the stability and immunogenicity of a T =1 Human Papillomavirus (HPV) L1-protein Virus-like Particle (VLP). Prospects of computer-aided vaccine design will be discussed.

The crystal structure of satellite tobacco mosaic virus (STMV) has been solved by a combination of multiple isomorphous replacement and molecular replacement methods and refined at 2.9 A resolution to a conventional R-factor of 0.215. STMV, a T = 1 icosahedral virus, is the smallest whose structure has been determined. The coat protein is an eight-stranded "Swiss roll" beta-barrel with an amino-terminal strand that extends away from the beta-barrel by more than 60 A. This strand is primarily responsible for quaternary interactions within the capsid. The most arresting feature of the virus structure is the intimate association of each capsid protein dimer with a Watson-Crick base-paired segment of RNA double helix on the interior of the virion. The icosahedral 2-fold axis of each dimer pair is coincident with that of the central base-pair of each helical RNA segment whose helical axis is along the edge of the icosahedron. The helical RNA segments are seven base-pairs in length with a stacked base at each 3' end so that a total of 16 nucleotides is clearly visible. The character of the RNA helix is somewhat different than any of the canonical forms. Assuming full occupancy, then approximately 45% of the total RNA genome is present in the electron density map. The close association of capsid with highly structured nucleic acid suggests that assembly of STMV is likely to be a highly co-operative process involving both protein and RNA. The nucleic acid is distributed within the virion with a high degree of order. The capsid protein is a true double helical RNA binding protein and a number of prominent interactions between protein and RNA can be clearly seen.

Quasi-elastic light scattering was used to investigate the nucleation and crystallization of apoferritin, ferritin and satellite tobacco mosaic virus (STMV). Supersaturation dependent critical nuclear size supportive of stable macromolecular crystal growth was estimated to be in the range of 5 to 65 monomer protein and virus particles. The interfacial free energy and the activation energy for apoferritin crystal growth was estimated to be 2.7 x 10 -2 erg/cm 2 and 8.5 kcal/mol, and for STMV 1.8 x 10 -2 erg/cm 2 and 10 kcal/mol, respectively. The data suggests that the rate limiting factor in STMV crystal growth is the diffusion of virus clusters to the growing crystal nucleus, while apoferritin crystallization is limited by surface kinetics. The aggregation rate is substantially higher for crystallization with a power law exponent of n = 0.33-0.54 (STMV) and n = 0.8-1.0 (apoferritin), while that associated with eventual amorphous precipitate was n = 0.20.

Quasi-elastic light scattering (QELS) was used to investigate homogeneous and heterogeneous nucleation events in the crystallization of satellite tobacco mosaic virus (STMV). Under conditions of relatively moderate supersaturation (σ ≈ 1.00–1.40), the critical nuclear size supportive of stable crystal growth, Rc, was estimated from time dependent size distribution analyses to be in the range of 30 to 7 virus particles respectively. From the same data, the molar interfacial free energy and the activation energy for crystal growth was deduced to be 2.2 and 10 kcal/mol, respectively. These, we believe represent the first such estimates for virus crystal growth. The acceleration of nucleation and crystal growth by the introduction of heterogeneous nucleants (latex microspheres) was demonstrated.

In plants, transgenes containing Potato spindle tuber viroid (PSTVd) cDNA sequences were efficient targets of PSTVd infection-mediated RNA-directed DNA methylation. Here, we demonstrate that in PSTVd-infected tobacco plants, a 134 bp PSTVd fragment (PSTVd-134) did not become densely methylated when it was inserted into a chimeric Satellite tobacco mosaic virus (STMV) construct. Only about 4–5% of all cytosines (Cs) of the PSTVd-134 were methylated when flanked by satellite sequences. In the same plants, C methylation was approximately 92% when the PSTVd-134 was in a PSTVd full length sequence context and roughly 33% when flanked at its 3′ end by a 19 bp PSTVd and at its 5′ end by a short viroid-unrelated sequence. In addition, PSTVd small interfering RNAs (siRNAs) produced from the replicating viroid failed to target PSTVd-134-containing chimeric STMV RNA for degradation. Satellite RNAs appear to have adopted secondary structures that protect them against RNA interference (RNAi)—mediated degradation. Protection can be extended to short non-satellite sequences residing in satellite RNAs, rendering them poor targets for nuclear and cytoplasmic RNAi induced in trans.

Satellite tobacco mosaic virus (STMV) is a small plant virus that is dependent for its replication on the presence of a helper tobamovirus. RNase protection analysis of genomic RNA of the STMV type strain revealed that it was composed of two major genome types which differed at a single detectable site near nucleotide 753. Analyses of 42 full-length STMV clones for sequence heterogeneity resulted in the identification of 16 variants distinguishable by unique RNase protection assay patterns. Characterization of these variants confirmed the presence of a major heterogeneity site at nucleotide 751 and identified several sites of sequence micro heterogeneity typical of an RNA quasispecies population. Mapping of the heterogeneity sites revealed an apparently random distribution along the length of the STMV genome, with no significant clustering or preference for noncoding regions. Infectivity experiments in tobacco showed that RNA transcripts of 13 of the 16 variant clones were infectious, indicating that most of the variants represent functional genomes coexisting in the type strain with the two major genome types. Sequence analyses revealed that most of the heterogeneity sites detected, including the major site of heterogeneity, were single base differences. Assessment of all the heterogeneity sites found in the total of 10,545 nt sequenced allowed us to estimate that the RNase protection assays detected approximately 50% of the differences present in the 16 clones studied. The nature of these differences was highly biased in that 18 of the 29 single base differences characterized (62%) were G to A substitutions.

This unit describes the use of satellite tobacco mosaic virus (STMV) vectors in combination with native TMV particles for inducing transient gene silencing in tobacco plants. Target gene fragment selection and insertion, virus delivery procedures, and phenotype screening of silenced plants are described in detail. All critical parameters for tobacco plant cultivation, virus infection, and RNA silencing efficiency are discussed.

Helical fibers, presumably proteinaceous and of microbial origin, have been visualized by atomic force microscopy on the surfaces of crystals of satellite tobacco mosaic virus. If the crystals are growing, then the fibers are incorporated intact into the crystal lattice. If broken on the crystal surface, then within a few minutes, the fibers self-reassemble to reestablish continuity. This, we believe, is the first observation of such a crystal surface–catalyzed repair of a biological structure. The surfaces of virus crystals provide ideal workbenches for the visualization and manipulation of nanoscale objects, particularly extended structures such as these fibers. Proteins 2001;44:392–396. © 2001 Wiley-Liss, Inc.

Retroviral Gag proteins direct the assembly and release of virus particles from the plasma membrane. The budding machinery consists of three small domains, the M (membrane-binding), I (interaction), and L (late or “pinching-off”) domains. In addition, Gag proteins contain sequences that control particle size. For Rous sarcoma virus (RSV), the size determinant maps to the capsid (CA)-spacer peptide (SP) sequence, but it functions only when I domains are present to enable particles of normal density to be produced. Small deletions throughout the CA-SP sequence result in the release of particles that are very large and heterogeneous, even when I domains are present. In this report, we show that particles of relatively uniform size and normal density are released by budding when the size determinant and I domains in RSV Gag are replaced with capsid proteins from two unrelated, nonenveloped viruses: simian virus 40 and satellite tobacco mosaic virus. These results indicate that capsid proteins of nonenveloped viruses can interact among themselves within the context of Gag and be inserted into the retroviral budding pathway merely by attaching the M and L domains to their amino termini. Thus, the differences in the assembly pathways of enveloped and nonenveloped viruses may be far simpler than previously thought.