Nucleotide sequence and translation of satellite tobacco mosaic virus RNA

Satellite tobacco mosaic virus (STMV) is a plant virus with a 17-nm icosahedral particle encapsidating a 0.3 X 10(6) Mr 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 [gamma-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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Liposome-mediated infection of plant protoplasts with tobacco mosaic virus RNA

Tobacco mosaic virus (TMV) RNA was encapsulated in large unilamellar vesicles (LUV) of phosphatidylserine and was introduced into protoplasts from Vinca rosea suspension cultures. Infection could be effected by brief incubation of protoplasts with the LUV containing TMV-RNA in the presence of either polyethylene glycol or polyvinyl alcohol. The presence of these polymers was essential for infection, and postinoculation washing with the high pH-high Ca2+ buffer enhanced infection significantly. Up to 80% of protoplasts could be infected under the optimal conditions as demonstrated by immunofluorescence technique. Calculations showed that around 2 x 10(6) TMV-RNA molecules were added per infected protoplast, indicating that the efficiency of infection by this method compares favorably with those by the existing methods for inoculating protoplasts from mesophyll cells with TMV-RNA. The significance of using protoplasts from cultured cells for infection with plant viruses and their nucleic acids is discussed.     

De novo emergence of a novel satellite RNA of cucumber mosaic virus following serial passages of the virus derived from RNA transcripts

Satellite RNA (satRNA) is often associated with cucumber mosaic virus (CMV); however, its origin remains unexplained and a subject for speculation. We passaged progeny of molecularly cloned CMV-Fny and CMV-LS in Nicotiana tabacum cv. Ky 14 under greenhouse conditions. A satRNA emerged after at least eight successive transfers of CMV-Fny, but no satRNA was recovered after eleven serial transfers of CMV-LS under the same conditions. The sequences of the newly emerged satRNA were determined, and an infectious cDNA clone was synthesized. Comparison of the sequences of the newly emerged satRNA with those of known CMV satRNAs showed that it is unique. This observation raises interesting questions regarding the enigmatic nature of the origin of CMV satRNAs. The nucleotide sequence data reported in this article have been deposited in the GenBank under accession number DQ975201.     

Structural transitions of satellite tobacco mosaic virus particles

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 A 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 A), the enlarged 18-nm particles crystallize in a cubic form which diffracts to no better than 5 A. 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.     

Characterization of a satellite RNA associated with cucumber mosaic virus

Two types of RNA, each with a molecular weight of approximately 0.12 × 10⁶, designated RNA 5 and satellite RNA, have been found in purified cucumber mosaic virus (CMV) preparations and have been characterized by molecular hybridization analysis using ³²P-labeled complementary DNA probes transcribed from these RNAs. RNA 5 usually makes up about 5% or less by weight of the total viral RNA and was shown to consist of specific cleavage products of CMV RNAs 1–4. Its nucleotide complexity was equivalent to about three times its molecular weight. By contrast, satellite RNA could form up to about 50% by weight of virion RNA and had the following properties: (1) It contained a unique nucleotide sequence with no homology with CMV RNAs, (2) CMV and tomato aspermy virus, but not alfalfa mosaic virus or tobacco ringspot virus, could function as helper viruses for its replication and encapsidation, (3) its nucleotide sequence was independent of the host plant and the helper virus used for its propagation and it was not derived from a host plant RNA, and (4) it was not a negative copy of any of the CMV RNA species. We concluded that this RNA is a true satellite RNA and has no relationship to the RNA found in defective interfering particles of animal viruses.     

Investigation of RNA structure in satellite panicum mosaic virus

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.     

Specific interaction of tobacco mosaic virus RNA with Q beta-replicase

The activity of Qβ-replicase is selectively inhibited by tobacco mosaic virus (TMV) RNA, while other RNA polymerases are not inhibited by TMV-RNA. Partially stripped TMV (PSV) is also effective in inhibiting Qβ-replicase.     

Cotranslational disassembly of tobacco mosaic virus in vitro

Heterogeneous nucleoprotein preparations of tobacco mosaic virus (TMV), stored at pH 7.0, were treated briefly at pH 8 and then incubated in mRNA-dependent rabbit reticulocyte lysate. The treatment at pH 8.0, under conditions which did not cause detectable polar disassembly as judged by exposure to micrococcal nuclease, caused TMV to stimulate in vitro translation up to 100-fold over background. High-molecular-weight products, characteristic of TMV, appeared, albeit at a significantly slower rate than when naked TMV RNA was used as template. Ribonucleoprotein material from cycloheximide-treated incubations was examined in the electron microscope to reveal a subpopulation of rodlets (approx 5%) with clusters of ribosomes at their concave 5' ends. It is proposed that pH 8 treatment removes the last turn of protective coat protein subunits from the rods and "exposes" at least a 49-nucleotide segment of the efficient 5'-leader sequence of TMV RNA for interaction with 40 S ribosomal subunits. Ribosome translocation during protein synthesis presumably dislodges further coat protein subunits progressively while continuing protection of the incoming TMV RNA.