Symptom-modulating properties of peanut stunt virus satellite RNA sequence variants.

The symptom-modulating properties of three peanut stunt virus (PSV) satellite RNA (satRNA) sequence variants were studied. The (V)-satRNA did not affect symptom development in tobacco plants infected with PSV. The (G)- or (WC)-satRNA, on the other hand, attenuated the symptoms. In these plants, the symptoms of PSV were restricted primarily to the inoculated leaves, and in some cases, a few leaves above the inoculated leaf showed small chlorotic areas. Northern blot analysis of total nucleic acids from PSV-infected plants containing the (V)-satRNA revealed the presence of both satellite and viral RNAs in inoculated leaves as well as in systemically infected leaves. On the other hand, satellite and viral RNAs were detected in the inoculated but not in the noninoculated leaves from infected plants containing either (G)- or (WC)-satRNA. Although a decrease in the quantities of genomic RNAs 1, 2, and 3 was characteristic of all satRNA-containing plants, this effect was more evident in the case of (G)- and (WC)-satRNAs. The complete nucleotide sequences of the three satRNAs were determined and compared to the published sequence of PSV satRNA. The (V)-satRNA differed from the published sequence at two positions, whereas the (G)- and (WC)-satRNAs differed at six and eight positions, respectively. Comparison of the nucleotide sequence of the satRNA having no effect on PSV-induced symptoms with those reducing virus symptoms suggests that a single nucleotide change or as many as five nucleotide changes may distinguish between attenuating and nonattenuating satRNAs.     

Integration of hepatitis B virus DNA in chromosome-specific satellite sequences.

We previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. We report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite DNA were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence.     

Evolution and replication of tobacco ringspot virus satellite RNA mutants.

The replication properties of linker insertion-deletion mutants of tobacco ringspot virus satellite RNA have been studied by amplification in plants infected with the helper virus. Sequence analysis of the cDNAs corresponding to the replicated forms shows that only one of the original mutated molecules replicates unaltered, and in general new variants accumulate. Depending on the location of the original mutation three types of sequence modifications were observed: (i) deletion of the mutated region followed by sequence duplication, (ii) sequence duplication and deletion outside of the mutated region and (iii) limited rearrangements at the site of mutation. The mutant that replicates without sequence changes accumulates linear multimeric forms suggesting that self-cleavage is affected although the sequence alteration does not involve the hammerhead catalytic domain. Alternative RNA conformations are likely to play a role in the origin of this phenotype and in the formation of sequence duplications. These results demonstrate the great structural flexibility of this satellite RNA.     

The 3'' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.     

Defective interfering RNAs of a satellite virus

Panicum mosaic virus (PMV) is a recently molecularly characterized RNA virus with the unique feature of supporting the replication of two subviral RNAs in a few species of the family Gramineae. The subviral agents include a satellite RNA (satRNA) that is devoid of a coding region and the unrelated satellite panicum mosaic virus (SPMV) that encodes its own capsid protein. Here we report the association of this complex with a new entity in the RNA world, a defective-interfering RNA (DI) of a satellite virus. The specificity of interactions governing this four-component viral system is illustrated by the ability of the SPMV DIs to strongly interfere with the accumulation of the parental SPMV. The SPMV DIs do not interfere with PMV satRNA, but they do slightly enhance the rate of spread and titer of PMV. The SPMV-derived DIs provide an additional avenue by which to investigate fundamental biological questions, including the evolution and interactions of infectious RNAs.     

The arginine-rich motif of Bamboo mosaic virus satellite RNA-encoded P20 mediates self-interaction, intracellular targeting, and cell-to-cell movement

Satellite RNA of Bamboo mosaic virus (satBaMV) has a single open reading frame for a nonstructural, RNA-binding protein, P20, which facilitates the long-distance movement of satBaMV in Nicotiana benthamiana. Here, we elucidate various biological properties of P20 and the involvement of a single domain in its activities. P20 displayed a strong self-interaction in vitro and in vivo, and cross-linking assays demonstrated its oligomerization. Domain mapping, using the bacterial two-hybrid system, indicated that the self-interacting domain overlaps the RNA-binding domain in the N-terminal arginine-rich motif (ARM) of P20. The deletion of the ARM abolished the self-interaction of P20 in vitro and in vivo and impaired its intracellular targeting and efficient cell-to-cell movement in N. benthamiana leaves. Moreover, RNA and protein accumulation of the ARM deletion mutant of satBaMV was significantly reduced in leaves systemically coinfected with Bamboo mosaic potexvirus and satBaMV. This is the first report of the involvement of ARM in various biological activities of a satellite RNA-encoded protein during infection of its host.     

Cytopathology of satellite tobacco mosaic virus and its helper virus in tobacco

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.     

The Subviral RNA Database: a toolbox for viroids, the hepatitis delta virus and satellite RNAs research

Background  

Viroids, satellite RNAs, satellites viruses and the human hepatitis delta virus form the 'brotherhood' of the smallest known infectious RNA agents, known as the subviral RNAs. For most of these species, it is generally accepted that characteristics such as cell movement, replication, host specificity and pathogenicity are encoded in their RNA sequences and their resulting RNA structures. Although many sequences are indexed in publicly available databases, these sequence annotation databases do not provide the advanced searches and data manipulation capability for identifying and characterizing subviral RNA motifs.     

The capsid protein of satellite Panicum mosaic virus contributes to systemic invasion and interacts with its helper virus

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and spread in host plants. The SPMV RNA encodes a 17-kDa capsid protein (CP) that is essential for formation of its 16-nm virions. The results of this study indicate that in addition to the expression of the full-length SPMV CP from the 5'-proximal AUG start codon, SPMV RNA also expresses a 9.4-kDa C-terminal protein from the third in-frame start codon. Differences in solubility between the full-length protein and its C-terminal product were observed. Subcellular fractionation of infected plant tissues showed that SPMV CP accumulates in the cytosol, cell wall-, and membrane-enriched fractions. However, the 9.4-kDa protein exclusively cofractionated with cell wall- and membrane-enriched fractions. Earlier studies revealed that the 5'-untranslated region (5'-UTR) from nucleotides 63 to 104 was associated with systemic infection in a host-specific manner in millet plants. This study shows that nucleotide deletions and insertions in the 5'-UTR plus simultaneous truncation of the N-terminal part of the CP impaired SPMV spread in foxtail millet, but not in proso millet plants. In contrast, the expression of the full-length version of SPMV CP efficiently compensated the negative effect of the 5'-UTR deletions in foxtail millet. Finally, immunoprecipitation assays revealed the presence of a specific interaction between the capsid proteins of SPMV and its helper virus (PMV). Our findings show that the SPMV CP has several biological functions, including facilitating efficient satellite virus infection and movement in millet plants.     

Identification of regions affecting virulence, RNA processing and infectivity in the virulent satellite of turnip crinkle virus.

Turnip crinkle virus (TCV) supports a small family of satellite RNAs (RNAs C, D and F). RNA C is a virulent satellite, producing severe symptoms in host plants, while RNAs D and F are avirulent satellites. The virulent satellite (RNA C) has two major domains--a 5'-domain similar to the avirulent satellites and a 3'-domain similar to the 3'-end of the TCV genome. To demonstrate that the 3'-domain of RNA C determines virulence, a chimeric satellite was constructed composed mostly of the 5'-domain of the avirulent satellite (RNA F) and the 3'-domain of the virulent satellite (RNA C). To locate other functional regions, small DNA fragments were inserted or deleted at various sites in the cDNA of virulent satellite (RNA C). Most small internal deletions and insertions in the midsection of the molecule had no detectable effects while those near the 3'-end of RNA C destroyed infectivity. Modifications in a small region centering on an AGCAGC repeat in the domain of satellite homology blocked the accumulation of monomers and presumably the processing of RNA C. Other modifications in this region produced more intense symptoms. Hence, these experiments reveal regions of the satellite which determine virulence, are essential for infectivity, affect monomer accumulation (RNA processing) and modulate symptom expression.     

The subunit structure of mouse satellite chromatin.

Atomic coordinates are presented for the 3740 atoms other than hydrogen in the dimeric molecule of chicken muscle triose phosphate isomerase. They are derived from an electron-density map at 2.5Åresolution, interpreted in terms of the known amino-acid sequence, and they have been adjusted systematically to give stereochemically appropriate bond lengths and angles.     

The primary structure of bovine satellite 1.715.

The primary structure of the 1402 bp repeat unit of bovine satellite 1.715 has been determined using a dimer inserted at the SalI site of plasmid pBR322 and cloned in E. coli. In contrast with bovine satellites 1.706, 1.720b and 1.711a, the 1.715 satellite has a complex sequence with no obvious internal short prototype repeat. The sequence consists however of repeats ranging in length from 6 to 13 nucleotides. In addition, the hexanucleotide, AGATGA, present in the prototype sequences of satellites 1.706, 1.720b and 1.711a, is found in satellite 1.715 in repeats as long as, or longer than, 8 nucleotides, establishing a homology link among those satellites on one hand and satellite 1.715 (and the related satellite 1.711b) on the other. In turn, this suggests a common evolutionary origin. A comparison of the maps for 15 restriction enzymes of cloned and uncloned satellite indicates very little sequence divergence among the repeat units of the latter, most of the differences being due to methylation.     

Plant virus satellite RNAs and their role in engineering resistance to virus diseases

The mechanism of satellite RNA (Sat-RNA) mediated attenuation of virus disease was suggested to be the result of competition between satellite RNAs and their helper viral RNAs for replication. Subcellular distribution of viral coat protein in cucumber mosaic virus (CMV) infected leaf tissues shows that the presence of Sat-RNAs interferes with the movement of CMV coat protein into chloroplants. As a strategy for controlling virus diseases, the expression of Sat-RNAs in transgenic plants confers some extent of resistance that may not be strong enough to protect the plants from natural virus infections. Transgenic plants expressing both the Sat-RNA and the coat protein of CMV exhibit enhanced resistance to the virus.     

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.     

Preliminary analysis of crystals of satellite tobacco mosaic virus

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 A 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 A, and it is probably 5 to 10 A less than this value.