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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.