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Satellite tobacco mosaic virus (STMV) can undergo at least two physical transitions that significantly alter its mechanical and structural characteristics. At high pH the 17-nm STMV particles expand radially by about 5 Å to yield particles having diameters of about 18 nm. This pH-induced transition is further promoted by aging of the virions and degradation of the RNA, so that swollen particles ultimately appear even at neutral pH. While the native 17-nm particles crystallize as orthorhombic or monoclinic crystals which diffract to high resolution (1.8 Å), the enlarged 18-nm particles crystallize in a cubic form which diffracts to no better than 5 Å. In the transition, not only do the capsid protein subunits move radially outward, but the helical RNA segments with which they interact do as well. This is noteworthy because it demonstrates that the RNA and the protein shell are capable of coordinated movement, and that neither structure is rigidly defined or independent of the other. Using atomic force microscopy, it can be shown that STMV particles, upon drying, lose their mechanical rigidity and undergo deformation. Virions initially 17 nm in diameter shrink to more uniform final sizes than do 18 nm, initially swollen particles. This transition appears to be irreversible, as the particles do not reassume their former size nor structural rigidity upon rehydration. Evidence is also presented that preparations of native virus and their crystals are naturally somewhat heterogeneous and contain a variety of particles of anomalous size. © 2001 Academic Press.

Biological macromolecules and particularly viruses, provide excellent systems for the study of crystallization from solution because of their relatively large size. The kinetics of their crystallization is at least an order of magnitude less than for conventional systems, and their large size permits visualization, both of crystal lattices and individual particles, by techniques such as atomic force microscopy (AFM). This technique is especially powerful for biological macromolecules because it can be utilized in situ, in the crystallization mother liquor, over long periods of time without perturbing the growing crystals. We present here observations using AFM of the nucleation and growth of crystals of satellite tobacco mosaic virus, and some recordings as well of bromegrass mosaic virus. Correlations are made, where possible, with corresponding analyses using X-ray diffraction analysis.

Highly purified virions of satellite tobacco mosaic virus (STMV) were found to crystallize at relatively low concentrations (300-500 microg ml(-1)) in pure water. Small crystals of these preparations were examined in the transmission electron microscope after either being rotary shadowcast with metal or negatively stained with 4% uranyl acetate. Stereo views were also obtained of both types of preparations. Stereo pairs of metal-coated crystals provided good three-dimensional images. When stereo pairs of negatively stained crystals were printed from second negatives, they provided striking images although the three-dimensional aspect was not so pronounced. Images of both types of preparations were compared with a computer-generated model of the virus. This model was based on data obtained in earlier X-ray diffraction crystallographic studies. Measurements of crystal axes on the EM images were somewhat lower than those of the computer model. It is assumed the reason for this is the dehydration of crystals during preparation for electron microscopy. The EM images did verify the type of crystal lattice determined in the X-ray diffraction studies. Conversely, knowing the exact unit cell parameters and the distribution of virions in the crystal from X-ray diffraction data aids in the further interpretation of electron micrographs of virus crystals.

Two crystal forms, orthorhombic and cubic, of satellite tobacco mosaic virus have been investigated. Atomic force microscopy showed that the orthorhombic crystals were characterized by a high density of point defects, while the cubic crystals were practically defect-free. Nonetheless, orthorhombic crystals diffract to a high resolution of 1.8 Å while the cubic crystals diffract to only about 4 to 6 Å resolution. Differences in the properties of viruses incorporated into the two crystal structures were demonstrated by growth kinetics studies. It appears that physical and chemical treatments applied to protein and virus solutions during their extraction and purification introduce a variety of specific structural changes and that these alterations may then affect the diffraction properties of resultant macromolecular crystals.

Satellite viruses contain particles and genomes that have structural and genetic features which indicate a viral origin but have lost (or never had) the ability to replicate by themselves in susceptible plant hosts. They lack an identifiable gene for a replicase of their own and so, in order to replicate, they need to be a dependent partner in a mixed infection with another virus, usually referred to as the helper virus. The helper virus is presumed to supply the replicase function the satellite virus lacks, but it is not needed for particle formation, since the satellite virus codes for its own capsid protein.

Nearly 40 years ago, Kassanis and Nixon (1960) described two kinds of particles in tobacco necrosis virus (TNV) infections that differed in size, and thus seemed to be separate viruses. The smaller component was demonstrated to depend on the larger autonomous virus for its multiplication, and hence was designated a satellite virus, satellite tobacco necrosis virus (STNV). This discovery quickly stimulated research in several areas of biology and chemistry with a focus on relationships, pathology, transmission, and interdependence of STNV and TNV. Following the realization of the satellite nature of STNV, identification of other satellite viruses was remarkably slow compared with the rates of discovery of new viruses, satellite RNAs, and viroids. More than 15 years passed before a second satellite virus, satellite panicum mosaic virus (SPMV; Fig. 1), was described (Buzen et al. 1984; Niblett and Paulsen 1975). A satellite virus particle associated with maize white line mosaic virus (MWLMV) that constitutes a third distinct satellite virus was subsequently identified (Gingery and Louie 1985; Zhang et al. 1991a). The helper viruses that support replication of these three satellite viruses are all isometric and have monopartite genomes. However, particle morphology of the helper virus seems to be inconsequential for support of isometric satellite viruses, since a fourth satellite virus, satellite tobacco mosaic virus (STMV), naturally occurs in Nicotiana glauca infected with the rod-shaped tobacco mosaic virus (Valverde and Dodds 1986).

Satellite tobacco mosaic virus replication, recombination and symptom modulation properties of the satellite RNAs of turnip crinkle virus biology and structure of plant satellite viruses activated by icosahedral helper viruses defective and defective interfering RNAs of monopartite plus-strand DNA plant virus human hepatitis delta virus structure and functional relationships of satellite RNAs of cucumber mosaic virus encapsidated circular viroid-like satellite RNAs of plants large satellite RNA RNA luteovirus-associated viruses and subviral RNAs.

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.

Atomic force microscopy (AFM) images at the molecular level have been obtained for a number of different protein and virus crystals. They can be utilized in some special cases to obtain information useful to crystal structure analyses by x-ray diffraction. In particular, questions of space group enantiomer, the packing of molecules within a unit cell, the number of molecules per asymmetric unit, and the dispositions of multiple molecules within the asymmetric unit may be resolved. In addition, because of the increasing sensitivity and resolution of the AFM technique, some molecular features of very large asymmetric units may be within reach. We describe here high-resolution studies, using AFM, to visualize individual molecules and viruses in their crystal lattices. These investigations included fungal lipase, lysozyme, thaumatin, canavalin, and satellite tobacco mosaic virus (STMV).

Evidence from a variety of space experiments confirms that gravity does, in some cases, have a significant effect on the crystallization of macromolecules. This has been demonstrated by numerous investigators employing a wide range of instruments and proteins. In the absence of gravity, convective transport of solute is eliminated and pure diffusion predominates. This has the consequence of producing local regions of reduced supersaturation thereby enhancing conditions for optimal growth. In addition, sedimentation of microcrystals and large impurities onto the surfaces of growing crystals, as well as direct contact between crystals, is minimized. The mechanisms for biological macromolecule crystallization, the unique features of their growth, and techniques for growing and characterizing crystals of proteins and viruses are described. The instruments currently available and under development for macromolecular crystal growth and its study in microgravity are reviewed.

The growth of six protein and virus crystals was investigated in situ using atomic force microscopy. Most of the crystals grew principally on steps generated by two-dimensional nucleation on surfaces though some grew by development of spiral dislocations. Apoferritin grew by a rarely encountered mechanism, normal growth, usually associated only with melt or vapour phase crystallization. Cubic crystals of satellite tobacco mosaic virus (STMV) grew, at moderate to high levels of supersaturation, by the direct addition of three-dimensional nuclei followed by their rapid normal growth and lateral expansion, a mechanism not previously described to promote controlled and reproducible crystal growth from solutions. Biological macromolecules apparently utilize a more diverse range of growth mechanisms in their crystallization than any previously studied materials.

For the first time, virus crystal growth dynamics and morphology have been investigated in real time on the nanometer scale. Individual monomers on the (111) face of cubic satellite tobacco mosaic virus (STMV) crystals were resolved and used to determine crystal packing. Growth of STMV proceeded by two- and three-dimensional nucleation to formed ``stacks'' of islands. No dislocations were observed. Small islands provided an estimate of critical radius size and the free energy of the step edge, \ensuremath{\alpha}. Step advancement rates were used to determinate the kinetic coefficient \ensuremath{\beta}. Images illustrate mechanisms for defect incorporation and suggest factors that limit growth rate and uniformity.

A series of frameshift and deletion mutations was created in the genome of satellite tobacco mosaic virus (STMV) by modifying full-length cDNA clones of the type strain, from which biologically active transcripts could be synthesized in vitro . Deletions and frameshift mutations in the 5′ open reading frame had no effect, compared to wild-type STMV, on RNA accumulation, systemic movement, or the symptoms induced by STMV in Nicotiana tabacum co-inoculated with tobacco mild green mosaic tobamovirus (TMGMV). This implies that the protein encoded by this reading frame is not necessary for biological activity. Deletions and frameshift mutations in the coat protein open reading frame resulted in decreased accumulation of STMV RNA in N. tabacum , although these mutants were still capable of systemic movement, presumably in a nonencapsidated or free RNA form. Furthermore, the mild symptoms induced in tobacco by co-inoculations of wild-type STMV/TMGMV or infection with TMGMV alone were altered to severe systemic necrosis when plants were co-inoculated with these STMV coat protein mutants and TMGMV. Mutants within the 3′ untranslated region were much less able to accumulate in TMGMV-infected plants than was wild-type STMV, and under some growth conditions did not accumulate to detectable levels.

The evolution of surface morphology and step dynamics during growth of single crystals of the protein Canavalin and of the cubic satellite tobacco mosaic virus crystals (STMV) have been investigated 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 cluster 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 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.

A model of secondary structure is proposed for the 3'-terminal sequence of the satellite tobacco mosaic virus (STMV) RNA on the basis of phylogenetic comparisons with tobacco mosaic virus (TMV) genomic RNA. Sequence homologies and compensatory base changes found between the two related viral RNAs imply that the 3'-end of STMV RNA folds into a tRNA-like domain similar to that found in the TMV RNA. Accordingly, functional assays showed that STMV RNA can be aminoacylated in vitro with histidine by yeast histidyl-tRNA synthetase to plateaus reaching 30%. Histidylation properties of STMV RNA were compared to those of TMV RNA and of a canonical yeast tRNA(His) transcript which both are chargeable to nearly 100% plateau levels. Kinetic data indicate an excellent catalytic efficiency of STMV RNA charging expressed as Vmax/Km ratio, quasi-equivalent to that of TMV RNA, and only 17-fold reduced as compared to that of the yeast tRNAHis transcript. Biological implications of the structural mimicry between the tRNA-like regions of TMV and STMV RNAs are discussed in the light of the relationships of a satellite virus with its helper virus. This is the first report on a chargeable tRNA-like structure at the 3'-end of a satellite virus RNA.

The type strain of satellite tobacco mosaic virus (STMV) contains two major variants, designated type 5 (T5) and type 6 (T6), which can be easily distinguished by RNase protection analyses. Clones containing cDNA of representative T5 and T6 STMV genomes have only five single-base differences in the entire 1059-nucleotide genome, and RNA transcribed from each clone is highly infectious when inoculated onto tobacco plants. The different RNase protection assay patterns can be used as genetic markers to identify individual STMV variants and to follow the interactions of variants and their progeny during coinfections in plants. The study described here investigated the effects of coinoculation and various delayed inoculations of T5 and T6 variants on the composition of the progeny STMV populations in systemically infected tobacco tissues. When T5 and T6 STMV RNAs were coinoculated or inoculated with 1-hr delays, the progeny from individual plants most often contained a mixture of T5 and T6 genomes. However, when there was a 24-hr delay between inoculations, the balance of T5 and T6 components in the progeny populations shifted toward predominance of the first variant inoculated. With delays of 3 or 7 days only the first variant was evident in the progeny populations, indicating that established replication of one STMV variant interferes with replication of another in a manner similar to the cross protection phenomenon.

We investigate the assembly of Satellite Tobacco Mosaic Virus (STMV) in a coarse-grained model. We use multi-level coarse-grained representations to decrease the computational expenses and adequately represent the different parts of the viral structure. The RNA coarse grain model was generated from a combination of an idealized RNA secondary structure based on the X-ray crystal and a proposed tRNA-like secondary structure at the 3’ end. The RNA model has one pseudo atom (bead) per residue. The coarse-grained model for the capsid contains 20 triangular units, each of which also contains three flexible positively charged protein tails. The assembly process as well as the stability of the virus mainly depends on RNA-protein and protein-protein interactions. The protein tails are attracted to the RNA by electrostatic interactions while the capsid proteins are weakly attracted with each other by hydrophobic interactions. We modeled RNA-protein interactions with a Debye-Huckel potential and protein-protein interaction with a Lennard-Jones potential. We varied values of these two interactions to find regions where the virus is stable and will self-assemble, and construct a phase diagram of viral stability. Finally we investigated the assembly of the virus using molecular dynamics. These simulations help us understand the individual roles of these two interactions on viral assembly.

The molecular basis of the interactions between plant virus satellites and their helper viruses is not understood. The features of the satellite tobacco mosaic virus (STMV) genome that determine tobamovirus helper specificity were investigated using two independent strategies. The first tested the possible significance of regions of nearly identical sequence within the 3′- terminal 150 bases of the genomes of STMV and its natural helper virus, tobacco mild green mosaic virus (TMGMV). A chimeric STMV clone containing the 3′ terminus of tobacco mosaic virus (TMV-U1) RNA was infectious in coinfections with TMGMV, but it did not replicate with TMV-U1. In the second strategy, populations of STMV adapted to replication with four alternative helper tobamoviruses were generated by serial passage in tobacco. RNase protection analyses of these RNA populations showed that in all cases there had been a genetic change 50 to 60 bases from the 5′ terminus of the STMV genome. Similar changes were detected in several progenies of STMV clones replicated with TMV-U1, indicating that change at this site was essential for replication with a helper virus other than TMGMV. Sequence analyses of the changes at this ‘helper adaptation domain’ showed consistently the deletion of a single G from five consecutive Gs at bases 61 to 65. Infectivity experiments with STMV clones containing this G deletion showed that this change alone was not sufficient to modify helper specificity, so additional factors which remain to be identified must also be involved. Nevertheless, these experiments show the ability of STMV populations to undergo rapid, reproducible evolution by both selection of pre-existing variants and de novo mutation, and constitute the first molecular demonstration that the helper virus acts as a selection pressure on the evolution of satellite populations.

Fifteen field isolates of satellite tobacco mosaic virus (STMV) were collected from wild Nicotiana glauca plants within a 2O-km2 area in southern California. Analyses of the entire length of the satellite genomic RNA of each isolate by RNase protection assays showed that there were 10 different genotypes within the 15 isotates, including some with regions of extreme divergence from the STMV type strain. This indicates a very high level of genetic diversity between natural STMV populations. Isolates from adjacent plants (within 3 m) were not always identical, but were more similar than those from more distant locations. Mapping of the genomic sites of heterogeneity revealed that in all cases the 3′ half of the genome, which comprises the untranslated region, was more conserved than were the 5′ coding regions.

Satellite tobacco mosaic virus (STMV) is the spherical satellite to an obligatory rod-shaped helper tobacco mosaic virus (TMV), which is required for replication. STMV has 60 protein subunits of M(r) 17,500 on a T = 1 icosahedral capsid containing a single-stranded RNA genome of 1,059 bases. STMV appears similar to another virus, STNV, but is approximately 20 per cent smaller. It shows no amino-acid homology or immunological cross-reactivity with either STNV or its host TMV. Here we report the X-ray crystal structure of STMV, which shows that the coat protein of STMV contains a 'Swiss roll' beta-barrel. An amino-terminal strand extends more than 60A and is primarily responsible for quaternary interactions. Each capsid dimer is associated with a segment of genomic RNA double helix comprising seven base pairs. The dyad of each protein dimer is coincident with that of the central base pair of the associated RNA segment whose helix axis is directed along an icosahedral edge. Protein-nucleic acid interactions are extensive. The RNA helices, which have additional stacked bases at their 3' termini, differ significantly from canonical nucleic acid helical forms.

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory — 1 (IML-1), flown January 22–29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20–22 °C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.

Recombinant cDNA clones of the complete satellite tobacco mosaic virus (STMV) genome (1059 ribonucleotides) were constructed with unique Xbal and HindIII or Pstl restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of T7 or SP6 phage promoters. Genome-sense RNAs transcribed in vitro from the T7 promoter were biologically active, while negative-sense RNAs transcribed in vitro from the SP6 promoter were not. Constructs that were identical to STMV and two other constructs in which there were two or six specific nucleotide differences in the 3' noncoding region yielded RNAs that were infectious. Sequence analysis of the progeny RNA derived from infections with transcripts containing nucleotide differences between nucleotides 682 and 753 revealed that these changes in sequence were maintained. In contrast, differences in the nucleotide sequence between nucleotides 989 and 1059 were not maintained in progeny RNA; one mutant reverted to the wild-type sequence, and the other generated a new sequence during infection.

Structures of the nucleoprotein of three negative strand RNA virus families, borna disease virus, rhabdovirus and influenza A virus, are now available. Structural comparisons showed that the topology of the RNA binding region from the three proteins is very similar. The RNA was shown to fit into a cavity formed by the two distinct domains of the RNA binding region in the rhabdovirus nucleoprotein. Two helices connecting the two domains characterize the center of the cavity. The nucleoproteins contain at least 5 conserved helices in the N-terminal domain and 3 conserved helices in the C-terminal domain. Since all negative strand RNA viruses are required to have the ribonucleoprotein complex as their active genomic templates, it is perceivable that the (5H+3H) structure is a common motif in the nucleoprotein of negative strand RNA viruses.

The presence of satellite tobacco mosaic virus (STMV) in coinfection with eight helper viruses in 23 hosts has previously been reported to cause no change in the symptoms induced by the helper viruses alone. This is not the case for systemic infection of certain types and cultivars of pepper, Capsicum annuum. In Jalapeno and Pimiento pepper plants, the presence of STMV in coinfections with TMV-U2 caused increased chlorosis in the form of abundant, bright-yellow patches relative to the mosaic caused by TMV-U2 alone. Increased chlorosis also was observed when TMV-U5 and pepper mild mottle virus (PMMV) were used as helper viruses [...]

Satellite tobacco mosaic virus (STMV) was detected frequently in California in association with TMV-U5 in naturally infected plants of Nicotiana glauca. Replication of STMV was not helper virus specific because eight tobamoviruses were able to support its replication in experimental infections in hosts that included crop plants such as tobacco and tomato. Two other tobamoviruses as well as several taxonomically distinct viruses could not serve as a helper. Infection with STMV did not alter symptoms induced by the helper viruses in any of the hosts tested (...)

Summary Isolates of tobacco mosaic virus strain U5 (TMV-U5) from native Nicotiana glauca plants induced the accumulation of a dsRNA (mol. wt. 0.6 × 106) in infected plants that was more abundant than the replicative form (RF) dsRNA of TMV (mol. wt. 4.3 × 106). Some but not all subcultures of such a field isolate obtained from single local lesions on N. tabacum cv. Xanthi-nc, had lost the ability to induce the 0.6 × 106 mol. wt. dsRNA. Co-inoculation experiments in N. silvestris established that the dsRNA could accumulate in plants infected with TMV-U1, but to a lesser extent than when associated with TMV-U5. A ssRNA (mol. wt. 0.3 × 106) was isolated from plants containing the dsRNA. This was not infectious by itself but became so when associated with TMV-U5 or TMV-U1, and then induced the accumulation of the 0.6 × 106 dsRNA. Plants infected with TMV-U5 isolates which did or did not induce the 0.6 × 106 dsRNA had identical symptoms. The host range of the 0.3 × 106 ssRNA was the same as that of the TMV strain with which it was associated in each of the 20 experimental hosts tested. Complementary DNA transcribed from purified 0.6 × 106 dsRNA did not hybridize with RF and other dsRNAs of TMV-U5, TMV-U1, tobacco necrosis virus, potato virus X, citrus tristeza virus, and cucumber mosaic virus + CARNA 5, but the cDNA did hybridize with the 0.6 × 106 mol. wt. dsRNA and the 0.3 × 106 mol. wt. ssRNA found only in plants containing this dsRNA. The results indicate that the 0.6 × 106 dsRNA is the RF of a satellite RNA of TMV. Purified nucleoprotein from plants infected with TMV-U5 and the satellite RNA were infectious for the satellite RNA, but the nature of encapsidation of the satellite RNA has yet to be determined.

Summary A ssRNA, which has a satellite-like relationship with tobacco mosaic virus (TMV), had an estimated mol. wt. of 0.38 × 106 and was found to be encapsidated in isometric particles with a diameter of 17 nm. The capsid protein had a mol. wt. of 18 × 103. This satellite virus of TMV (STMV) is highly immunogenic and no serological relatedness was detected between it and either TMV or satellite tobacco necrosis virus (STNV) or satellite panicum mosaic virus (SPMV) in immunodiffusion tests with antibodies specific for STMV, TMV, STNV and SPMV. Randomly primed STMV cDNA hybridized with STMV-specific dsRNA and ssRNA but did not hybridize to ssRNA of TMV, SPMV, panicum mosaic or cucumber mosaic viruses. The dsRNAs of different isolates of STMV differed in their electrophoretic mobilities, but they all shared nucleotide sequence homology. Particles of STMV tended to aggregate and crystallize upon purification. Replication of STMV in tobacco plants was supported by TMV but not by cucumber mosaic virus. This is the first report of a satellite virus of a rod-shaped plant virus.

The relatedness of the genomes of satellite panicum mosaic virus (SPMV) and its helper virus, panicum mosaic virus (PMV), were investigated by nucleic acid hybridization. The results show that the satellite and helper virus RNAs have no appreciable homology or complementarity as assessed by hybridization with cDNA probes derived from the genomes of PMV and SPMV and with a probe complementary to the 3′ terminus of SPMV RNA. The complete nucleotide sequence of SPMV RNA reveals that the genome is 826 nucleotides (nt) long. The ability to label SPMV RNA with polynucleotide kinase only after phosphatase treatment suggests that the 5′ terminus is phosphorylated, but the extent of phosphorylation was not determined. The first open reading frame (ORF), encountered after an 88-nt 5′-untranslated region, encodes a 17,000 mol wt protein of a size and amino acid composition that are consistent with analysis of SPMV coat protein. An additional short ORF, located near the 3′ end of the RNA, could encode a 6300 mol wt polypeptide. The minus strand also contains two ORFs that could potentially encode polypeptides of 7100 and 11,000 mol wt. No evidence is available to determine whether the second positive-strand ORF or the two minus-strand ORFs are expressed. The data presented here clearly show the SPMV RNA is distinct from the RNAs of other satellite viruses, in both size and nucleotide sequence. However, the 5′-untranslated portions of SPMV and satellite tobacco mosaic virus RNAs share some structural features that may be important in initiation of translation.

Ultrastructural responses of tobacco cells infected with a newly discovered satellite virus (STMV) that has an isometric morphology and is associated with rigid rodshaped tobacco mosaic virus (TMV) were studied in situ . In cells infected with TMV alone,TMV particles occurred as crystalline arrays in the cytoplasm and were usually associated with TMV-characteristic X bodies. In cells infected with both TMV and STMV, particles of STMV occurred only in cells that contained TMV particles, which suggests a correlation between the satellite and helper virus presence. However, the replication and/or accumulation sites of STMV appear to be independent from its helper virus. Unlike TMV particles, STMV particles were associated with several cytopathic structures such as granular inclusions, membranous vesicles of 50–80 nm, and myelin-like bodies which were all bounded by a single common membrane, No X bodies occurred in cells containing STMV particles, and the mitochondria possessed abnormal tubular structures containing flocculent material.

In this study, we assembled an Agrobacterium-based transient expression system for the ectopic expression of Satellite tobacco mosaic virus (STMV) (+) or (−) transcripts and their biological activity was confirmed when Nicotiana benthamiana plants were co-expressed with helper Tobacco mosaic virus replicase. Characterization of STMV in the presence and absence of its HV revealed: (i) HV-dependent expression of STMV (+) in N. benthamiana, but not in N. tabacum, generated a replication-deficient but translation and encapsidation competent variant lacking the highly conserved 3′ 150 nucleotides (nt) (STMVΔ150); (ii) mutational analysis demonstrated that a conserved 3′ stem-loop structure in wild type and STMVΔ150 located between nt 874 and 897 is essential for translation of CP; (iii) helper virus-independent expression of CP from wt STMV was competent for the assembly of empty aberrant virion-like particles; whereas, CP translated from STMVΔ150 resulted in disorganized CP aggregates suggesting a role for the 3′tRNA-like structure in STMV assembly.

SummaryThis work presents an all-atom molecular dynamics simulation of a complete virus, the satellite tobacco mosaic virus. Simulations with up to 1 million atoms for over 50 ns demonstrate the stability of the entire virion and of the RNA core alone, while the capsid without RNA exhibits a pronounced instability. Physical properties of the simulated virus particle including electrostatic potential, radial distribution of viral components, and patterns of correlated motion are analyzed, and the implications for the assembly and infection mechanism of the virus are discussed.

Viral genomic RNA adopts many conformations during its life cycle as the genome is replicated, translated, and encapsidated. The high-resolution crystallographic structure of the satellite tobacco mosaic virus (STMV) particle reveals 30 helices of well-ordered RNA. The crystallographic data provide global constraints on the possible secondary structures for the encapsidated RNA. Traditional free energy minimization methods of RNA secondary structure prediction do not generate structures consistent with the crystallographic data, and to date no complete STMV RNA basepaired secondary structure has been generated. RNA-protein interactions and tertiary interactions may contribute a significant degree of stability, and the kinetics of viral assembly may dominate the folding process. The computational tools, Helix Find & Combine, Crumple, and Sliding Windows and Assembly, evaluate and explore the possible secondary structures for encapsidated STMV RNA. All possible hairpins consistent with the experimental data and a cotranscriptional folding and assembly hypothesis were generated, and the combination of hairpins that was most consistent with experimental data is presented as the best representative structure of the ensemble. Multiple solutions to the genome packaging problem could be an evolutionary advantage for viruses. In such cases, an ensemble of structures that share favorable global features best represents the RNA fold.

Viruses are compact biological nanoparticles whose elastic and dynamical properties are hardly known. Inelastic (Brillouin) light scattering was used to characterize these properties, from microcrystals of the Satellite Tobacco Mosaic Virus, a nearly spherical plant virus of 17-nm diameter. Longitudinal sound velocities in wet and dry Satellite Tobacco Mosaic Virus crystals were determined and compared to that of the well-known protein crystal, lysozyme. Localized vibrational modes of the viral particles (i.e., particle modes) were sought in the relevant frequency ranges, as derived assuming the viruses as full free nanospheres. Despite very favorable conditions, regarding virus concentration and expected low damping in dry microcrystals, no firm evidence of virus particle modes could be detected.

Satellite tobacco mosaic virus (STMV), a small T = 1 icosahedral plant virus, has been crystallized in a form suitable for high-resolution X-ray diffraction analysis. The crystals, which diffract to better than 2.5 Å resolution, are of space group I222 and have unit cell dimensions of a = 176 A ̊, b = 192 A ̊ and c = 205 A ̊. The centers of the virus particles occupy 222 symmetry points in the unit cell and one quarter of the virus particle constitutes the asymmetric unit, which is therefore comprised of 15 capsid protein molecules. From packing considerations, the maximum diameter of the STMV particles cannot exceed 165 Å, and it is probably 5 to 10 Å less than this value. © 1989.

Satellite tobacco mosaic virus (STMV) is a plant virus with a 17-nm icosahedral particle encapsidating a 0.3 × 106Mr ssRNA genome that depends on tobamoviruses for its replication. The complete nucleotide sequence of STMV RNA deduced in the experiments described here was 1059 nucleotides in length. The efficiency of labeling viral RNA with [γ-32P]ATP using T4 polynucleotide kinase was not affected by treatment with tobacco acid pyrophosphatase and/or bacterial alkaline phosphatase, indicating that the majority of the 5′ termini of encapsidated STMV RNAs were not phosphorylated. The 240 3′-terminal nucleotides of STMV RNA and either tobacco mosaic virus (TMV) U1 RNA or TMV U2/U5 RNA had greater than 65% overall sequence similarity, with two nearly identical regions of 40 and 50 bases, respectively. There were no other regions of sequence relatedness to TMV RNA. The 19 5′-terminal nucleotides of STMV RNA had greater than 65% sequence similarity with the 16 5′-terminal nucleotides of brome mosaic virus (RNA 3 and 50% sequence similarity with the 12 5′-terminal nucleotides of the Q strain of cucumber mosaic virus RNA 3. The first open reading frame (ORF) beginning at base 53 encoded a 6800 Mr protein that corresponded in size to a major in vitro translation product directed by STMV RNA. A second ORF, beginning at nucleotide 163, had the capacity to code for a protein that corresponded in size (17,500 Mr) to the other major in vitro translation product. The first 12 codons of this ORF corresponded to the sequence of the N-terminal amino acids of the capsid protein. Western-blot analysis of the in vitro translation products revealed that the 17,500 Mr protein had the same electrophoretic mobility as the authentic capsid protein; it was also antigenically related to the capsid protein, but the 6800 Mr protein was not. Time course analysis of in vitro translation demonstrated that the 6800 Mr protein was synthesized at the same time as the capsid protein and did not arise by the proteolytic cleavage of a larger precursor polypeptide. These results suggest that the genome of STMV functioned as a polycistronic messenger RNA. It has not been determined if the 6800 Mr protein is synthesized in vivo. STMV RNA had untranslated regions of 52 and 418 nucleotides at its 5′ and 3′ termini, respectively. Nonphosphorylated 5′ termini, the degree of similarity to the 3′ terminus of two of its helper viruses, the genome organization, and the ability to function as a polycistronic mRNA are unique features for the genome of this satellite virus.

Most investigations on biological macromolecules are important for the knowledge of the functions in living organisms. Nowadays it is well known that the three-dimensional structure of proteins is obtained either by NMR or X-ray crystallographic methods. The crucial part in the latter is the availability of high-quality crystals in order to perform structural characterization. Nevertheless, there are some approaches to overcome this problem from the statistical [Jancarik and Kim, J. Appl. Crystallogr. 24 (1991) 409] and physico-chemical point of view [Ries-Kautt and Ducruix, Methods in Enzymology 276 Part A, Ch. 3, 1997, p. 23]. Once the crystals are obtained, the following part of the research must be focused on growing the crystal in order to have an appropriate size for X-ray analysis. There are additional advances in the methods for crystallizing, growing and determining what kind of biophysical or biochemical parameters have to be taken into account in order to obtain a high quality protein crystal, these advances have been already published elsewhere [Ducruix and Giege, Crystallization of Nucleic Acids and Proteins. A Practical Approach, IRL Press, Oxford, 1991; McPherson, The Preparation and Analysis of Protein Crystals, Wiley, New York, 1982]. In order to evaluate these parameters, we have developed a new technique, called the gel acupuncture technique for crystallizing proteins inside an X-ray capillary tube as well as for trying to study the “in situ” crystal growth phenomena [Garcia-Ruiz et al., Mater. Res. Bull. 28 (1993) 541; Garcia-Ruiz and Moreno, Acta Crystallogr. D 50 (1994) 484]. In this work, we present our recent investigations on the influence of the gravity vector upon protein crystallization. Three proteins were chosen in order to test this possible influence, taking into account the size of each: satellite tobacco mosaic virus (1000 kDa) and two proteins of “low molecular weight”, thaumatin I (22 kDa) and concanavalin A (200 kda). The experimental set-up was the same for the gel acupuncture technique [Garcia-Ruiz et al., Mater. Res. Bull. 28 (1993) 541] and the experiments were carried out ranging from 0° (parallel) to 180° (anti-parallel) to the gravity vector to check its influence on protein crystallization. Thus, the counter-diffusion phenomena in protein crystallization were evaluated from parallel, 45°, 90° up to anti-parallel transport of the protein molecules to the gravity vector. Finally, we discussed why the molecular weight of the protein system should be taken into account in order to avoid a sedimentation phenomena and how the crystal growth technique is determined in order to keep the same diffusion pattern throughout the experiment.