Albert Siegel

Replication of tobacco mosaic virus: I. Isolation and characterization of double-stranded forms of ribonucleic acid

Abstract Two species of double-stranded RNA (DS RNA) were isolated from TMV-infected leaf tissue with the aid of cellulose chromatography. They have characteristics of replicative form (RF) and replicative intermediate (RI) found in other RNA virus infected tissue. Their buoyant densities in Cs 2 SO 4 are, respectively, 1.615 and 1.630 g/ml, whereas TMV-RNA has a buoyant density of 1.640 g/ml. DS RNA displays a sharp thermal transition at 68° in 10 −3 M potassium phosphate buffer containing 10 −3 M Na 2 EDTA and is detected in appreciable amount only in a cell fraction sedimenting with mitochondria. RF and RI have estimated molecular weights, respectively, of 4 × 10 6 and 5.0 × 10 6 daltons as determined by polyacrylamide gel electrophoresis. DS RNA is not infectious but becomes so upon thermal denaturation. It is probably composed of TMV-RNA and its complementary strand on the basis of the above evidence and because tritium-labeled TMV-RNA becomes partially resistant to nuclease action upon incubation with denatured DS RNA under annealing conditions.

Replication of tobacco mosaic virus: III. Viral RNA metabolism in separated leaf cells☆☆☆

Abstract The metabolism of RNA has been studied in cell suspensions prepared from tobacco mosaic virus (TMV)-infected tobacco leaves. The cell suspensions incorporated [ 3 H]-uridine into encapsidated viral RNA at a constant rate for as long as 44 hr. Gel electrophoretic analyses of RNA extracted from infected and uninfected cells which had been exposed to [ 3 H]uridine, showed that both incorporated label into ribosomal RNA species. However, RNA preparations from infected cells contained five additional species of RNA not observed in preparations from healthy cells; in addition to TMV RNA, label was detected in two species of double-stranded RNA identified as replicative form (RF) and replicative intermediate (RI), a single-stranded RNA component of low molecular weight (LMC) and one with a molecular weight greater than that of TMV RNA. Synthesis of all five species of virus-specific RNA was insensitive to actinomycin D indicating their independence of cellular DNA. Cell suspensions prepared from infected leaves, when pulsed for short periods with [ 3 H]uridine, incorporated isotope preferentially into R.I and RF. On the addition of excess unlabeled uridine, the radioactivity was chased completely from RI and partially from RF into TMV RNA, whereas there appeared to be no significant turnover in the isotope associated with LMC. Results of these studies suggest that RI and RF may be progenitors of TMV RNA; however, the inability to completely chase [ 3 H]uridine from RF raises additional possibilities, which are discussed.

In vitro synthesis of double-stranded RNA by an enzyme system isolated from tobacco leaves

Abstract An RNA-dependent RNA polymerase which catalyzes the polymerization of ribonucleotides into an acid-insoluble product has been isolated from tobacco leaves and partially characterized. 1. 1. The enzyme activity is found in the 30 900 × g supernatant fraction of a leaf homogenate. The reaction requires the presence of a divalent metal ion and four ribonucleoside triphosphates for activity. 2. 2. The product of the enzyme-catalyzed reaction is double-stranded RNA as evidenced by its ribonuclease resistance and sharp thermal transition at 80 °C in 30 mM Na+. It is of low molecular weight (less than 5 S) as determined by polyacrylamide gel electrophoresis. 3. 3. Addition of various species of RNA stimulates the polymerization reaction, but the added RNA does not appear to be associated with the enzyme product. It appears neither to act as a template nor as a primer. 4. 4. The enzyme can be isolated from apparently healthy plants, but is also found in tobacco mosaic virus infected plants; its specific activity increases after infection. The enzyme activity and its product are not altered by infection.

Replication of tobacco mosaic virus: IV. Further characterization of viral related RNAs

Abstract Experiments were performed to characterize the viral related RNA species which appear in extracts of tobacco mosaic virus (TMV)-infected tissue and, in particular, the low molecular weight (ca. 350,000 daltons) component, LMC. It was determined that LMC is probably not a component of the virus rod but is a fragment of unincapsidated TMV RNA. Synthesis of LMC in diseased tissue is not inhibited by the presence of actinomycin D. Because LMC would not reconstitute into a rod with TMV protein, it was considered not to contain a detectable amount of the 5′ terminus of TMV RNA. Polyadenylic acid sequences could not be detected by three analytical methods in any RNA component. In addition to LMC, which is homogeneous in size, TMV RNA fragments polydisperse in size are also present in leaf tissue extracts. In contrast, RNA complementary to TMV RNA was present in extracts only as a component of the double-stranded TMV replicative form and was not found free in the infected cell. Neither fragments of replicative form nor single-stranded TMV complementary RNA could be detected. In addition, the only RNA fraction of uniform size found to contain both an RNA species and its complement was the TMV replicative form.

Pseudovirions of tobacco mosaic virus.

Abstract Tobacco mosaic virus (TMV) preparations contain a small percentage of pseudovirions. These particles, which are thought to consist of host RNA encapsidated with TMV protein, can be separated from virus particles by electrophoresis in 0.5% agarose gel. Pseudovirion RNA is smaller than and has a broader size distribution than viral RNA. It is complementary to host DNA, more so to chloroplast DNA than to nuclear DNA. Strains of TMV have different pseudovirion contents, with the U2 strain having the greatest proportion (2–2.5%) of the strains tested. U2 pseudovirion RNA is complementary to about 20% of tobacco chloroplast DNA and 0.4% of tobacco nuclear DNA. A large proportion of pseudovirion RNA is complementary to both chloroplast and nuclear DNA, suggesting that chloroplast DNA has many nucleotide sequences in common with nuclear DNA.

DNA Complementary to Ribosomal RNA: Relation between Genomic Proportion and Ploidy

Ten Nicotiana species were assayed for the proportion of DNA that is complementary to ribosomal RNA. This proportion varies from 0.27 to 0.9 percent, with tetraploid species having lower values than the diploid species. The tetraploid species have about twice as much DNA per cell as do diploid species. Thus, the absolute number of genes for ribosomal RNA varies less than the proportion of complementary DNA. Further, the number of genes for the RNA in 80S ribosomes varies less among species than does that for the RNA in 70S ribosomes. The data indicate that loss of DNA complementary to ribosomal RNA is associated with tetraploidy in the genus Nicotiana.

A STUDY OF NECROTIC LESION FORMATION BY TOBACCO MOSAIC VIRUS.

Abstract TMV strains U1 and U2 multiply in Nicotiana sylvestris , but only strain U2 induces local lesion formation that is accompanied by an enhanced oxygen consumption. The local lesions and the tissue surrounding them appear to be rich in polyphenoloxidase; lesions on leaves treated with various polyphenols darkened markedly within a few hours and a lesser darkening of the interlesion areas followed. In addition, polyphenols accelerated the appearance of necrotic infective centers by about 12 hours on U2-infected leaves, but had no observable effect on leaves infected with strain U1. Ascorbic acid reduced lesion numbers; lesions which did form were larger and less pigmented than those in the controls. This study supports and extends the conclusions of others who have indicated that the phenomenon of necrotic lesion formation involves polyphenols and polyphenoloxidase; however, it is felt that additional factor(s) are also involved.

A temperature-sensitive mutant of TMV with unstable coat protein☆

Abstract A temperature-sensitive mutant of TMV with a functional defect in the coat protein was isolated after nitrous acid treatment of the common strain. At 23 ° infected plants contained characteristic nucleoprotein virions and crystalline inclusions composed of disks of viral coat protein. At 35 ° no such particles or inclusions were produced; only free viral RNA and insoluble viral protein were found. The coat protein isolated from the virus differed from common strain protein in that it was stable only in low ionic strength buffers at neutral pH, did not aggregate very readily into virus-like rods, and denatured readily when aggregated at low pH. The protein has two amino acid exchanges involving a change from threonine to alanine at position 81 and serine to phenylalanine at position 143.

Artificial production of mutants of tobacco mosaic virus.

Publisher Summary The chapter discusses on the methods and results of mutation studies that have been carried out with tobacco mosaic virus (TMV). Of the small viruses that are available, tobacco mosaic virus (TMV) seems particularly well suited for study of the relationship between gene and gene product. The approach that has employed (TMV), and which has yielded valuable information, has been to modify chemically the viral nucleic acid and to study the consequent perturbations, which occur in the viral protein, the nucleic acid can be modified by treating it with mutation-inducing agents whose action is well understood. The effects of mutation on one of the gene products of TMV RNA have been intensively studied. This gene product is the readily available virus protein. Two types of alterations may occur to this protein as a result of mutation. The protein may be altered in amino acid composition but still remain functional, or the protein may become nonfunctional as a result of a changed amino acid composition. The appearance of the latter class of mutants demonstrates that the viral protein is not essential to the maintenance of infection. It is questionable, therefore, whether a lethal deamination can occur in that part of the viral nucleic acid concerned with specifying the viral protein. The typo of mr1tation, which results in an altered but still functional viral protein, is most intensively studied. All mutants of this class have their amino acid sequence altered in such a way that one amino acid replaces another at a particular position on the polypeptide chain. Most of the mutants of this class have a single amino acid replacement. A few mutants have two or three replacements, but these replacements are never found to be close to each other. This information provides strong evidence for a nonoverlapping genetic code.

Characterization of a new defective strain of TMV

Abstract A new nitrous acid-induced defective strain of tobacco mosaic virus (TMV) designated PM5 was isolated and some of its characteristics were studied. The coat protein of this defective strain showed a change from arginine to cysteine at position 112 in the amino acid sequence of the protein. The protein aggregated into rodlike structures similar to aggregated TMV protein but the subunits seem to be more loosely packed with a tendency to stacked disk configuration.