Immunocytochemical localization of the 130K and 180K proteins (putative replicase components) of tobacco mosaic virus

We have revealed the cellular localization of the putative replicase components of tobacco mosaic virus (TMV), 130K and 180K proteins, in TMV-infected tobacco leaves by the immunogold technique with antisera which specifically react with these two proteins. When sections of TMV-infected tobacco leaves were treated with anti-130K protein antiserum and then with protein A-gold complex, most of the gold label was strongly localized on granular inclusion bodies which were found specifically in the cytoplasm of TMV-infected cells. Very small amounts of label present in other regions, including the nuclei, chloroplasts, and mitochondria, seemed to be nonspecific. Gold-labeled 180K protein was also dispersed over the granular inclusion bodies. The granular inclusion bodies appeared to be oval-shaped structures with various diameters ranging from 0.2 to 2.8 microm. TMV particles were usually observed near the granular inclusion bodies as aggregates but not inside them. Considering the involvement of the 130K and 180K proteins in replication, the granular inclusion bodies may be the site for replication of TMV RNA.     

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

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.     

Studies upon the reaction of certain tobacco graft components against the tobacco mosaic virus

Summary Grafts between tobacco plants, chiefly such that show symptoms of tabacco mosaic virus (A-types) and other types, especially such that form necrotic lesions (B-types) were made and mutual reactions between scion and stock were studied after infection. Sometimes graftings were made between haelthy and infected graft-components. Several kinds of reactions were determined, which are interesting from a pathological and a developmental point of view, especially AB-reaction and BA-necrotic reaction. The former is manifested on the leaves of tobacco plants of the type A, while the latter on the young leaves of B-types in the infected graftings A-scion/B-stock or B-scion/A-stock. On the basis of the experimental data certain ideas are advanced, that may serve as working hypothesis.These experiments were completed in December, 1941, but technical causes prevented the publication.     

Homogenization-resistant and -susceptible components of tobacco mosaic virus replicative form RNA.

When prepared from tissue frozen with liquid nitrogen, tobacco mosaic virus replicative form RNA (TMV RF) was uniform in size but when prepared by high-speed homogenization, or when TMV RF prepared with liquid nitrogen was homogenized, 80 to 90% of the RF broke into relatively discrete pieces. The unbroken RF was not fragmented by additional homogenization. The TMV RF components susceptible and resistant to breakage, respectively, were synthesized with similar kinetics in relation to length of labelling period, but the slightly more resistant component was synthesized during the early infection period. Both components were produced by different strains of TMV but leaves infected with cowpea chlorotic mottle or southern bean mosaic viruses yielded only RF resistant to breakage. TMV replicative intermediate RNA was also broken by homogenization. The occurrence of the two RF components may be of significance in the replication of RNA viruses.     

Replication of alfalfa mosaic virus RNA: evidence for a soluble replicase in healthy and infected tobacco leaves

Soluble RNA-dependent RNA polymerases from healthy and alfalfa mosaic virus (AMV)-infected leaves were purified more than 400-fold from the 100,000g supernatant of leaf homogenates, using ammonium sulfate precipitation, Sephadex G-100 filtration, and chromatography on DEAE Sephadex A25 and P11-phosphocellulose. The DEAE-chromatography step eliminated host poly(U)polymerase-like activity, cellular RNases, and endogenous RNA, yielding a typical RNA-dependent RNA polymerase ("DEAE-enzyme") which was dependent on exogenous RNA. On nondenaturing 4-30% gradient slab gels, the activity of the enzyme was recovered in two peaks containing proteins of 340K and 160K. RNA products were synthesized in the presence of the "DEAE-enzyme" from healthy and infected leaves: When an excess of AMV RNA was introduced into the reaction mixture, only synthesis of minus strand RNA was detected. However, when limiting amounts of AMV RNA were used as template some small plus strands were detected, suggesting that synthesized minus strands may in turn serve as template. In both cases, the products were partially double stranded, and of very heterogenous size. Their size depended on the length of the RNA template which in turn varied according to the degree of RNase contamination of the enzyme fractions. These results are the first demonstration that both healthy and AMV-infected tobacco leaves contain a soluble replicase, although its possible in vivo role in viral replication is yet to be demonstrated.     

Turnip yellow mosaic virus RNA-replicase contains host and virus-encoded subunits

The enzyme (RNA-replicase) involved in the synthesis of viral RNA has been purified from turnip yellow mosaic virus (TYMV)-infected chinese cabbage leaves. The RNA-replicaset contains two major subunits: one of apparent molecular weight 115,000 (115K) and the other of 45K. We have raised antisera against the purified TYMV-RNA-replicase and have demonstrated by immunoaffinity chromatography and immunoblotting that the 115K polypeptide is coded by the viral RNA but that the 45K protein is of host origin. Furthermore the TYMV RNA-replicase is clearly different from the RNA-dependent RNA polymerase that occurs in healthy as well as in infected plants.     

Characterization of the virus encoded subunit of turnip yellow mosaic virus RNA replicase

An antiserum raised against TYMV-RNA encoded protein P115 partially inhibits TYMV RNA replicase activity, demonstrating that this protein is involved in TYMV RNA synthesis. The detection of protein P115 by an antibody linked polymerase assay demonstrates that protein P115 is indeed a subunit of the TYMV RNA replicase, the enzyme known to synthesize viral RNA in infected Chinese cabbage. The use of translation products of other tymoviruses indicates that the serological relationship between the virus-encoded replicase subunits of these viruses and protein P115 is very weak at the best.     

Evidence for intrastrand complementation in cowpea mosaic virus infection

In supplementation tests both middle component (M)-RNA and bottom component (B)-RNA of cowpea mosaic virus mutant N142 were shown to carry mutations affecting local symptoms in bean and cowpea. The M- and B-RNA mutations were separately incorporated into the two reciprocal hybrids of N142 and wild-type Sb, designated as M142BSb and MSbV142. Mixtures of the two hybrids induced normal local symptoms in bean and cowpea as a result of reassortment of components. This was also the case with a mixture of MSbV142 and mutant N123. The latter mutant was earlier shown to contain a M-RNA mutation for defective symptom expression in bean and cowpea. Although both M142BSb and N123 carried M-RNA mutations, mixtures of these isolates induced normal local symptoms. Subculturing showed no wild-type virus to be present in the inoculated leaves. Thus, intrastrand complementation was assumed to result in restoration of normal symptom production. We conclude that the results of genetic mixing experiments with multicomponent plant viruses must be interpreted cautiously. Subculturing experiments should be performed to determine whether restoration of wild-type symptoms results from exchange of nonmutated components or from intrastrand complementation.     

Differential pulse-polarographic analysis of tobacco mosaic virus

The tobacco mosaic virus (TMV) strain vulgare and its mutant TMV 483 (with glutamine-9 replaced by histidine) and the denatured protein of TMV vulgare were analysed by direct current (d. c.) and differential (derivative) pulse polarography (DPP) in the basic electrolyte composed of 0.001 M Co(NH3)6Cl3, 0.1 M NH4Cl and 0.1 M NH3 at 0 degrees C. The DPP method gave a substantially better resolution of the polarographic catalytic maxima A and B, but a much lower resolution of the maxima B and C, as compared with the d. c. polarographic method. The clear differentiation of the maximum A from maximum B by DPP permitted to study the variation of maximum A in the course of alkaline degradation of TMV. But for the study of TMV protein denaturation the d. c. polarography is preferable, because the denaturation is accompanied by the appearance and rise of maximum C which can be clearly differentiated from maximum B by d. c. polarography rather than by DPP. The DPP method was more, sensitive than d. c. polarography. The denatured TMV protein can be determined by DPP at concentrations around 0.1 microgram/ml.     

Infection of tobacco leaf epidermal protoplasts with tobacco mosaic virus

Protoplasts from the epidermal cells of tobacco leaves were prepared and inoculated with tobacco mosaic virus. Up to 50% of the protoplasts became infected and, within 3 days, contained an average of 5 x 10(5) virus particles per protoplast. Hexagonal crystalline inclusions were observed in many of the infected protoplasts.