Synthesis Of Tobacco Mosaic Virus RNA Research Articles

Tobacco mosaic virus replicase and replicative structures.

The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years. Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complementary RNA, initiated in vivo; however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template. The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase. Our recent studies have emphasized several aspects of TMV RNA replication. We have examined the nature of TMV replicative structures synthesized in vitro by utilizing a partially purified enzyme preparation isolated from TMV-infected tobacco tissue. Radiolabelled products of the reaction were analysed on agarose gels and fractions with the predicted electrophoretic migration and nuclease sensitivities of replicative form (RF) and replicative intermediate (RI) were isolated. These fractions were hybridized to a collection of bacteriophage M13 clones containing portions of the TMV genome of both plus and minus polarity. The nascent synthesis in the RI-like molecules was restricted to the plus viral strand, while the new synthesis in the RF-like molecules was of both plus and minus polarity. Solubilization of the membrane-bound replicase with the non-ionic detergent CHAPS has yielded complexes which remain in solution after high-speed centrifugation. The solubilized replication complexes have been utilized as starting material for enzyme purification by Sepharose 4B gel filtration chromatography. The intracellular site of synthesis of TMV RNA has been reinvestigated in the light of reports suggesting a nuclear site of replication. The conclusion for nuclear synthesis has been based on fractionation of subcellular homogenates of virus-infected leaves or mesophyll protoplasts and identification of virus-related proteins associated with these fractions. In our studies, however, we conclude that these procedures can be misleading in that the 126,000 Mr TMV protein (and replicase activity) were found in all fractions of the homogenate analysed. Double-stranded TMV RNA, on the other hand, was barely detectable in preparations of purified nuclei; instead it was concentrated in the post-nuclear supernatant, suggesting that the nucleus is not the site of TMV RNA synthesis.

Specific Inhibition of Tobacco Mosaic Virus Protein and Single-Stranded RNA Synthesis by Arabinofuranosyladenine

9-beta-D-Arabinofuranosyladenine (ara A) was shown to inhibit a specific step of tobacco mosaic virus (TMV) multiplication. The time course of the ara A-sensitive function occurred 'late' coinciding with the period of viral RNA and protein synthesis. When treatment began after viral RNA synthesis was established, 1.0 mM ara A inhibited viral single-stranded RNA and viral protein synthesis, but not viral double-stranded RNA synthesis. Viral double-stranded RNA synthesis was inhibited totally only when treatment with ara A began within the first 4 h (before any RNA synthesis was detected), and the degree of inhibition decreased rapidly when treatments began at later times. In contrast, single-stranded TMV RNA synthesis and viral protein synthesis were inhibited throughout the infection. The mode of action of ara A against TMV involves selective inhibition of a function required for single-stranded RNA synthesis and viral protein synthesis without detectable inhibition of RNA or protein synthesis of the host or ongoing viral double-stranded RNA synthesis.

Stimulation by aurintricarboxylic acid of tobacco mosaic virus-specific RNA synthesis and production of informosome-like infection-specific ribonucleoprotein

It was shown that aurintricarboxylic acid (ATA), a well-known inhibitor of protein synthesis, markedly stimulates the synthesis of tobacco mosaic virus (TMV)-specific RNA species of the intermediate (I) class (apparent molecular weights 1.1–1.3 × 10 6 and 0.6–0.8 × 10 6). No stimulation by ATA of full-length genomic TMV RNA or the subgenomic TMV RNA coding for TMV coat protein was detected. Informosome-like infection-specific ribonucleoprotein (vRNP) particles different from mature TMV particles were found in the TMV-infected cells (Yu. L. Dorokhov, N. M. Alexandrova, N. A. Miroshnichenko, and J. G. Atabekov, 1983, Virology 127, 237–252). It is shown here that [ 3H]uridine incorporation into vRNP RNAs was markedly stimulated in the presence of ATA. vRNP can be released from the TMV-specific polyribosomes by EDTA treatment, which suggests that it is involved in the translation process. The results of the pulse-chase experiments (including those in which TMV RNA synthesis is blocked by 2-thiouracil) suggest that vRNP does not serve as a precursor for mature virion.

Effects of 2-α-hydroxybenzylbenzimidazole on tobacco mosaic virus and host RNA synthesis in tobacco leaf discs and plants

2-α-hydroxybenzylbenzimidazole (HBB) at 3 μM strongly inhibited the accumulation of tobacco mosaic virus (TMV) particles over a 5-day-period, when applied soon after inoculation. TMV RNA synthesis was inhibited by up to 50% by HBB when leaf discs in which virus multiplication was well established were treated. HBB also inhibited host DNA, RNA and polyadenylated mRNA synthesis, at least as much as it inhibited TMV RNA synthesis. HBB was unable to prevent accumulation of TMV when applied to whole plants. The possible mode of action of HBB and its use in virus elimination methods are discussed.

Tobacco mosaic virus protein synthesis is correlated with double-stranded RNA synthesis and not single-stranded RNA synthesis

The synthesis rates of three proteins of tobacco mosaic virus (TMV), 160, 110, and 17.5 kDa, were monitored at intervals after interruption of synthesis of TMV RNA. Following inhibition of synthesis of both single-stranded and double-stranded RNAS by shifting wild type TMV to 40° or is mutant III 2-35 to 35°, the synthesis rates of viral proteins declined sequentially, with that of the larger proteins declining faster. When viral RNA synthesis was prevented with cordycepin, synthesis rates of the 110 and 160-kDa proteins declined rapidly, while the 17.5-kDa protein decreased more slowly. These data imply that the functional mRNA is transitory, probably nascent RNA, and that each protein is produced independently. The process of translation of viral mRNA was not temperature sensitive and occurred normally for brief periods after shift to restrictive temperatures. When single-stranded RNA synthesis was inhibited differentially from double-stranded RNA synthesis, protein synthesis was correlated with double-stranded RNA synthesis and not single-stranded RNA synthesis. Following a shift of is mutant IV-35 to 35°, a shift that immediately stopped single-stranded RNA synthesis without inhibiting double-stranded RNA synthesis, all three viral proteins continued to be produced normally. Also, after return of wild type TMV to 25° after a 1-hr incubation at 40°, viral protein and double-stranded RNA synthesis recovered in parallel to the normal rate after 8 hr whereas single-stranded RNA synthesis, which had been reduced more drastically, recovered more slowly after 16 hr.

Effect of 2-Thiouracil on RNA and Protein Synthesis in Synchronous and Asynchronous Infections of Tobacco Mosaic Virus

Examination of the effect of 2-thiouracil on tobacco mosaic virus (TMV) RNA and protein synthesis in synchronous and asynchronous systems of replication in tobacco leaves demonstrated that 2-thiouracil does not prevent synthesis of TMV RNA, as suggested by previous studies, but inhibits some earlier function. When added at different times after mechanical inoculation, 1 mM 2-thiouracil partially inhibited both viral RNA and protein synthesis, with greater inhibition when treatment began nearer the time of inoculation. In leaves systemically inoculated with TMV using a differential temperature inoculation procedure to synchronize the infection, 1 mM 2-thiouracil inhibited viral protein and RNA synthesis totally when treatment began within the first 4 h after initiation of replication, but not at all when treatment began at 12 h or later, even though earlier reports had shown that most RNA and protein synthesis occurred after 12 h.

Recovery of tobacco mosaic virus RNA Replication after incubation at 40 degrees.

Incubation of tobacco mosaic virus (TMV)-infected tissue at 40 degrees for 12 h destroyed the capacity for TMV RNA replication. Upon shift of the tissue from 40 to 25 degrees, synthesis of TMV RNA did not resume. Incubation at 40 degrees for 1 h did not destory the TMV RNA replicase, because viral RNA synthesis resumed upon return to 25 degrees. However, upon further incubation at 25 degrees, the synthesis rate of TMV RNA gradually declined. Upon still further incubation at 25 degrees (16-20 h), the synthesis of TMV RNA in tissue incubated at 40 degrees for either 1 or 12 h recovered. This recovery was inhibited by cycloheximide but not by 2-thiouracil.

Synthesis of TMV RNA at restrictive high temperatures

The primary effect of high temperatures that restrict tobacco mosaic virus (TMV) multiplication was an immediate inhibition of synthesis and a more gradual degradation of single-stranded TMV RNA. Incorporation of radioactive precursors into doublestranded TMV RNA, replicative form (RF) and replicative intermediate (RI), was affected much less. Immediately after a step-up shift from 25 to 40°, single-stranded TMV-RNA synthesis was almost totally prevented, whereas there was little inhibition of incorporation into RF or RI. However, incorporation of 32P into double-stranded TMV RNA rapidly declined after the synthesis of single-stranded TMV RNA had stopped. At high temperatures that were restrictive for TMV multiplication the function responsible for incorporation of 32P into single-stranded viral RNA was uncoupled from that responsible for incorporation into viral double-stranded RNA.

Guanidine inhibits tobacco mosaic virus RNA synthesis at two stages.

Guanidine (GD) inhibited tobacco mosaic virus (TMV) RNA synthesis at two different steps, depending on the concentration of GD used. The time-course of inhibition by 40 mM GD coincided with the time-course of RNA synthesis. However, 10 mM GD inhibited an earlier step that occurred prior to viral RNA synthesis.

Effect of cycloheximide on tobacco mosaic virus synthesis in callus from hypersensitive tobacco

Local lesions were formed on callus derived from Nicotiana tabacum cv Xanthi-nc 36–44 hr following inoculation with tobacco mosaic virus (TMV), but these had little or no inhibitory effect on virus synthesis. The greatest accumulation of [ 3H]-uridine- and [ 14C]leucine-labeled virus occurred 48–84 hr after inoculation. When infected callus tissue was treated with cycloheximide (1 μg/ml) during the period of rapid virus accumulation, the synthesis of host protein and complete virus was reduced by 90% within 1 hr. The synthesis of TMV RNA was not affected even after 4 hr of treatment. The results suggest that cycloheximide prevented the formation of complete virus by inhibiting the synthesis of viral protein and/or by preventing the assembly of virus particles.

The polarity of synthesis of the ribonucleic acid of tobacco mosaic virus

Tobacco mosaic virus-RNA was labeled with 33P for 90 min in Turkish tobacco plants in the presence of actinomycin D. The resulting product was extracted from the leaf tissue by treatment with 80% phenol and reconstituted with TMV-protein. Protein subunits were then stripped from the 3′-end of the RNA by treatment with sodium dodecyl sulfate. Digestion of the partially exposed RNA with pancreatic ribonuclease showed a concentration of 32P near the 3′-end. It is therefore concluded that the direction of synthesis of tobacco mosaic virus-RNA is from the 5′- to the 3′-end.

Double-stranded tobacco mosaic virus RNA

Isolation of the double-stranded viral RNA in fractionated cell-free extracts from radioactive tobacco mosaic virus-infected tobacco leaves suggests that the double-stranded tobacco mosaic virus RNA is associated with a cytoplasmic structure or organelle and not with nuclei. Synthesis of double-stranded viral RNA in cell-free extracts from infected leaves also occurred in association with cytoplasmic fractions and not with nuclear fractions. It is concluded that synthesis of tobacco mosaic virus RNA occurs in either mitochondria, in light chloroplasts lacking starch grains, or in some other unidentified sedimentable organelle. Evidence suggesting a nuclear site for tobacco mosaic virus RNA synthesis is discussed.