Milton Zaitlin

Replication of tobacco mosaic virus: V. Properties of the bound and solubilized replicase

Abstract Insoluble, bound tobacco mosaic virus (TMV) replicase, isolated from leaves of TMV-infected plants has been shown to catalyze the synthesis of nucleotide sequences of both TMV-RNA and its complement. Treatment of the complex containing the bound enzyme with Nonidet P-40 and KCl releases a TMV replicase which may then be further purified by sedimentation into a glycerol gradient. The partially purified enzyme is markedly stimulated by added RNA templates, but it exhibits little specificity for any of the 9 RNAs tested. The product of the partially purified enzyme was complementary to the RNA used as template and was bound to the template forming a ribonuclease-resistant, double-stranded molecule. The partially purified enzyme exhibits characteristics expected of an RNA-dependent RNA polymerase; viz., the requirement for four ribonucleoside triphosphates for maximum activity, insensitivity to added actinomycin D, rifampicin, deoxyribonuclease, and orthophosphate ion, sensitivity to added pyrophosphate or ribonuclease, and stimulation by RNA. The partially purified replicase cosediments in glycerol gradients approximately with human gamma globulin, suggesting it has a molecular weight of about 160,000.

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.

Replication of tobacco mosaic virus: II. The in vitro synthesis of high molecular weight virus-specific RNAs☆☆☆

Abstract The products synthesized in vitro by a crude replicase enzyme extract prepared from TMV-infected tobacco plants have been analyzed by polyacrylamide-agarose gel electrophoresis. The activity of this enzyme is insensitive to deoxyribonuclease and actinomycin D. About 65% of the phenol-extracted product is insensitive to pancreatic ribonuclease but is completely digestible by KOH. A portion of the synthesized RNA coelectrophoreses with double-stranded TMV replicative form of molecular weight of 4 × 10 6 daltons. A ribonuclease-sensitive, higher molecular product was detected on occasion; it was considered to be the TMV replicative intermediate of about 5 × 10 6 daltons. No single-stranded RNA corresponding in size to TMV-RNA was observed.

Unusual Aggregation of a Nonfunctional Tobacco Mosaic Virus Protein

The nonfunctional virus protein isolated from plants infected with the PM2 strain of tobacco mosaic virus aggregates to form elongated, two-stranded, open helical structures, in marked contrast with functional tobacco mosaic virus protein which aggregates into rods. This unique type of aggregation may explain why the PM2 protein is unable to combine with viral nucleic acid to form stable infectious virus particles.

Amino acid composition of a nonfunctional tobacco mosaic virus protein

Abstract The nonfunctional virus protein isolated from plants infected with the PM2 strain of tobacco mosaic virus aggregates in an aberrant manner and is unable to coat the virus nucleic acid effectively. It is similar in amino acid composition to the protein of the common strain with the exception that, of the 158 amino acids in the subunit, a threonine residue of the common strain has been replaced by isoleucine and a glutamic acid (or glutamine) residue has been replaced by aspartic acid (or asparagine) in PM2. The threonine-to-isoleucine change has been localized in the large N-terminal peptide (positions 1–41) obtained upon tryptic digestion of PM2 protein. The second replacement has not been localized, although it is not in the N-terminal peptide. Either one or both of these replacements is in a critical region concerned with the proper aggregation of the protein subunits into rods.

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.

Proteins in tobacco mosaic virus-infected tobacco plants

Abstract Tobacco mosaic virus (TMV)-infected leaf tissue was exposed to 3 H-leucine following a treatment with actinomycin D. Uninfected leaf tissue was similarly treated, but was labeled with 14 C-leucine. Extracts of the two tissues were combined and analyzed by electrophoresis in sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. At least four polypeptides were either engendered or stimulated in diseased tissue; one of these was the coat protein. Incorporation into one component was depressed in diseased tissue.