Translation of soybean mosaic virus RNA in vitro: evidence of protein processing

The genomic RNA of soybean mosaic virus (SMV), a member of the potyvirus group of plant viruses, was translated in both the wheat germ and reticulocyte cell free systems to identify some viral encoded proteins and as an approach to determining the translational strategy of the virus. The RNA was translated into the same specific set of 10 to 12 polypeptides in both in vitro systems. Immunological tests and peptide analyses indicate that six translation products are related to SMV coat protein, and one of these comigrated with coat protein during electrophoresis. Two other antigenically distinct classes of polypeptides were identified by their specific immunoprecipitation with antibody against the SMV cytoplasmic inclusion body protein or tobacco etch virus nuclear inclusion body protein. To determine if any products of the in vitro translation reactions resulted from proteolytic processing of a precursor molecule, translation reactions were carried out with amino terminal label N-formyl[35S]methionyl-tRNA(Met)i (f-Met), or with [35S]methionine, and the resultant products were compared. The putative SMV coat protein and a translation product related to the nuclear inclusion body protein were not labeled with f-Met indicating that they were generated by proteolytic processing at their amino termini. Consistent with this finding is the accumulation of new polypeptides of greater apparent molecular weight when amino acid analogs were present during translation.     

Further studies on the translation products of tobacco rattle virus RNA In vitro

Translation of RNA-1 (MW, 2.5 × 10⁶) of tobacco rattle virus (TRV) strain PRN in wheat germ extracts containing spermidine resulted in the production of many polypeptides with a maximum MW of 170,000 and a clear band of 140,000. Translation of RNA-1 in lysates of rabbit reticulocytes resulted in these two products but little else. PRN-RNA-2 (MW, 1.0 x 10⁶) was translated in both in vitro systems as two polypeptides, coat protein and a protein of MW 31,000 (band 1). Three bands of possible premature termination products were also produced in wheat germ extracts. The identity of coat protein was shown by coelectrophoresis in 10 and 3% polyacrylamide gels, specific aggregation with PRN protein, and coincidence between tryptic peptide fingerprints. Tryptic peptide mapping showed that band 1 was not a precursor for coat protein. There were, however, some peptides in digests of band 1 which coincided with coat protein peptides, which suggests a partial overlap of amino acid sequences. The relative abundance of coat protein and band 1 protein in translation products of PRN-RNA-2 in wheat germ extracts was dependent on the concentration of magnesium ion in the extracts. At concentrations suboptimal for total incorporation, translation resulted mainly in coat protein, whereas, at supraoptimal concentrations of magnesium, band 1 protein predominated in the translation products. We suggest that PRN-RNA-2 contains two open initiation sites.     

Translation of the four major RNA species of cucumber mosaic virus in plant and animal cell-free systems and in toad oocytes

Cucumber mosaic virus (CMV) contains four major RNA species, designated RNAs 14 in order of decreasing molecular weight. As a preliminary means of analysing gene content and distribution between these RNA species, each of the RNAs was purified and used to direct protein synthesis in cell-free systems from wheat embryos, wheat germ, and rabbit reticulocytes, and in intact oocytes of the toad, Bufo marinus. In vitro translation products of the CMV RNAs (Q strain) were analysed by electrophoresis on discontinuous slab polyacrylamide gels in the presence of sodium dodecyl sulphate. RNA 1 and RNA 2 proved untranslatable in oocytes but both directed synthesis of a large number of products in the wheat and rabbit reticulocyte cell-free systems. The highest molecular weight products obtained, about 105,000 for RNA 1 and 120,000 for RNA 2, represent most of the coding potential of these RNAs. Thus, RNA 1 and RNA 2 may be monocistronic messengers for the 105,000 and 120,000 MW products, respectively. Translation of RNA 3 in the wheat cell-free systems yielded three major products of molecular weights 34,000–39,000. In contrast, RNA 3 directed synthesis of essentially one product of molecular weight about 34,000 in both the rabbit reticulocyte system and oocytes. RNA 3 did not direct synthesis of coat protein in any of the systems, contrary to our earlier results and the known presence of the coat protein gene on RNA 3. RNA 4 directed synthesis of coat protein and higher- and lower molecular weight minor products in the cell-free systems and in oocytes. A product corresponding to the entire coding potential of RNA 4 was observed in the rabbit reticulocyte system, but not in the wheat systems or toad oocytes. The conflicting results obtained for translation of RNA 3 and RNA 4 in the different systems illustrate the danger in analysing genetic material by in vitro translation. The discussion considers which of the in vitro products are most likely to be synthesized in vivo and a tentative model of the CMV genome is proposed.     

Translational strategies in potexviruses: products encoded by clover yellow mosaic virus, foxtail mosaic virus, and viola mottle virus RNAs in vitro

We have examined the template properties of the genomic RNAs from three potexviruses: clover yellow mosaic virus (CYMV), foxtail mosaic virus (FMV), and viola mottle virus (VMV). All three RNAs encode a large (160,000 to 182,000 molecular weight) nonstructural protein when translated in the rabbit reticulocyte cell-free system. Only CYMV RNA is able to direct the synthesis of significant quantities of a product whose electrophoretic mobility, antigenic determinants, and partial peptide map resemble those of authentic coat protein. All three viral RNAs also encode a number of discrete polypeptide products whose molecular weights are intermediate between those of the large nonstructural proteins and those of their respective coat proteins. We have examined the kinetics of synthesis in vitro of these intermediate products. For each of the three viral templates, as well as for papaya mosaic virus RNA, pulse-chase experiments suggest that most, if not all, the intermediate polypeptides are incomplete ("paused") or prematurely terminated precursors to the corresponding large nonstructural protein. A partial proteolytic map of the in vitro products encoded by CYMV RNA supports this interpretation. Our data, and those of others, are consistent with a model in which all potexviruses would encode a large nonstructural protein.     

Protein synthesis in tobacco mesophyll protoplasts induced by tobacco mosaic virus infection

Tobacco mesophyll protoplasts inoculated in vitro with tobacco mosaic virus (TMV) were used to study protein synthesis induced or stimulated by virus infection. Ultraviolet irradiation of inoculated protoplasts sufficiently reduced host protein synthesis without markedly affecting TMV replication. In addition to viral coat protein, incorporation of amino acids into at least two high-molecular-weight proteins were demonstrated in infected protoplasts. One of these was unrelated to coat protein, consisted of a single polypeptide of about MW 140,000 and was present in association with some cellular structure less dense than nuclei or chloroplasts. The other protein with molecular weight of about 180,000 also appeared to be unrelated to coat protein, since it was labeled with amino acids not contained in the coat protein. Synthesis of the 140,000-MW protein followed a time course very similar to that of viral RNA replication and attained a maximum incorporation rate 4 hr earlier than coat protein synthesis. Production of progeny virus particles closely followed the course of coat protein synthesis.     

Fractionation of cucumber mosaic virus RNA and its translation in a wheat embryo cell-free system.

Cucumber mosaic virus (CMV) contains four major RNA species designated RNAs 1–4 in order of decreasing molecular weight. We report the preparative fractionation of CMV-RNA and the in vitro translation of the purified RNA species in a wheat embryo cell-free system.CMV-RNA was fractionated by 2–3 cycles of centrifugation in 10–40% sucrose density gradients employing a heat denaturation step after the first centrifugation. This method yielded a pure mixture of RNA 1 and RNA 2 (RNA 1 + 2), pure RNA 3 and pure RNA 4. The heat denaturation step was found necessary for elimination of hidden breaks and possible RNA aggregates in the final purified RNA samples.The purified CMV-RNA's were translated in a wheat embryo cell-free system in the presence of ³H-labeled amino acids and the ³H-labeled products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. With RNA 1 + 2 as messenger, four major and approximately eight minor products were found (molecular weight range 13,000–90,000). RNA 3 yielded CMV coat protein and one or two other products of molecular weight 26,000–28,000. Coat protein was the single translation product of both RNA 4 and unfractionated CMV-RNA. The identity of the ³H-labeled coat protein product was confirmed by cyanogen bromide cleavage and comparison of the peptides with those obtained from marker ¹⁴C-labeled coat protein.     

Translation of virus mRNA: synthesis of bacteriophage PP7 proteins in cell-free extracts from Pseudomonas aeruginosa

The translation of Pseudomonas phage PP7 RNA has been investigated, using cell-free extracts from Pseudomonas aeruginosa, strain 1. S-30 extracts translated PP7 phage RNA quite efficiently, but the isolated ribosomes incubated with an Escherchia coli S-100 supernatant preparation, were even more active. Three distinct products are made which correspond to in vivo labeled PP7 phage polypeptides judged by polyacrylamide-SDS gels. It is suggested that the PP7 genome has at least three cistrons.The major product of in vitro synthesis, synthesized in great excess of the other products, coelectrophoreses with authentic coat protein prepared from labeled phage. Tryptic peptide analysis of this product yielded leucine labeled tryptic peptides very similar to those of PP7 coat protein. It is concluded that this in vitro polypeptide is the product of the coat protein cistron. This product was also efficiently synthesized when PP7 RNA was translated by E. coli ribosomes, but the other PP7 genome products are not made, except occasionally in very small amounts. This result, and the fact that Pseudomonas ribosomes translate PP7 RNA more efficiently than R17 and Qβ RNA, indicates differential cistron recognition between Pseudomonas and E. coli ribosomes or their initiation factors.     

In vitro translation products of turnip crinkle virus RNA

Turnip leaves infected with turnip crinkle virus accumulate a 35-kd polypeptide which comigrates with the major protein from isolated virions. RNA from TCV virions directs the synthesis of a number of polypeptides in vitro including a 35-kd protein which is immunoprecipitable with antiserum to virus particles. Translation of viral RNA size-fractionated on sucrose gradients or methyl mercurichydroxide-containing gels shows that the TCV coat protein is synthesized primarily from RNA fragments which are smaller than the genomic RNA. A satellite RNA species found in virions does not direct the synthesis in vitro of any identifiable protein.     

Expression of the large 5'-proximal cistron of tobacco mosaic virus by 70 S ribosomes during cotranslational disassembly in a prokaryotic cell-free system

In vitro translation of pH 8-washed particles of tobacco mosaic virus (TMV; vulgare, common, or U1 strain) in a cell-free system derived from Escherichia coli (MRE 600) results in a broad size range of TMV RNA-encoded products, including significant amounts of the distinctive 126-kDa polypeptide which is encoded by the first, 5'-proximal open reading frame in TMV RNA and is conventionally expressed by 80 S eukaryotic ribosomes. In an identical prokaryotic translation system, unencapsidated TMV RNA encodes a series of polypeptides of 50 kDa or less. The predominant product of 16-17 kDa frequently appears to comigrate with TMV coat protein. The significance of the former result for mRNA-ribosome interactions and for possible virus-uncoating/early gene expression events within the chloroplasts of infected cells is discussed.     

The location of coat protein and viral RNAs of alfalfa mosaic virus in infected tobacco leaves and protoplasts

The location of coat protein of alfalfa mosaic virus (AIMV) strain 425 was determined in protoplasts isolated from infected tobacco leaves and in in vitro inoculated tobacco protoplasts, using immunocytochemistry on ultrathin frozen sections labeled with colloidal gold. In infected tobacco leaves 5 days postinoculation (p.i.) coat protein is present in the cytoplasm and nucleus, especially around the nucleolus. In in vitro inoculated tobacco protoplasts coat protein was not present in the nucleus 6 hr p.i. These results indicate that the presence of coat protein in the nucleus is not necessary for viral replication. However, coat protein could be detected in the nucleus 48 hr p.i. Probably coat protein or virus particles accumulate in the nucleus late in infection. Minus-strand RNA, as part of the replication complex, could be detected in a 650 g pellet fraction of infected tobacco leaves but not in the nucleus, suggesting that replication of AIMV occurs outside the nucleus.     

The proteins synthesized in tobacco leaves infected with tobacco necrosis virus and satellite tobacco necrosis virus

The synthesis of radioactively labeled proteins in extracts of infected leaves was studied by polyacrylamide gel electrophoresis in sodium dodecyl sulfate containing buffers. A double labeling technique and treatment of leaves with actinomycin D aided in distinguishing the synthesis of virus-induced or virus-stimulated proteins from the synthesis of host proteins.Six virus-induced or virus-stimulated proteins were detected in leaves infected with tobacco necrosis virus (TNV). In addition to the viral coat protein, proteins of molecular weights 64,000, 42,000, 23,000, 15,000 and 12,000 were resolved. The synthesis of the viral coat protein greatly exceeded that of the nonstructural proteins. The TNV genome is sufficiently large to code for all six proteins detected.Studies of extracts of leaves coinfected with satellite tobacco necrosis virus (STNV) and TNV revealed the synthesis of only one STNV-induced protein, the viral coat protein. When large amounts of STNV coat protein were synthesized, the production of the TNV coat protein of both strains A and B was selectively suppressed. The selective reduction of TNV coat protein synthesis suggested that STNV interference with TNV multiplication is effected at least in part at the translational level.     

Stable dimers of the coat protein of a strain of tobacco mosaic virus.

A large proportion of the coat protein of the tobacco mosaic virus, strain flavum, which accumulates in an insoluble form in infected tissue is present as a dimer. The dimer resists disaggregation by heating in the presence of sodium dodecyl sulfate, mercaptoethanol, and urea, and it cannot be completely dissociated by performic acid oxidation.     

Molecular weights of plant virus coat proteins by polyacrylamide gel electrophoresis

The molecular weights of the coat proteins of 15 plant viruses were estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most well-characterized viral coat proteins gave values within about 10% of that estimated by other means. Five viral proteins (cucumber mosaic virus, potato virus X, sowbane mosaic virus, tobacco etch virus, and white clover mosaic virus) gave values significantly different from those based on chemical and physical data. Two components were obtained with the coat proteins of tomato bushy stunt virus and white clover mosaic virus.     

Synthesis in vitro of the coat protein of papaya mosaic virus

Papaya mosaic virus (PMV) RNA directs the synthesis in vitro in either the wheat germ or reticulocyte lysate translation systems of numerous products. Three polypeptides are most prominent: pA (155,000 molecular weight), pB (73,000), and pC (22,000). The last, pC, displays the same molecular weight as coat protein and is precipitated by antibodies raised against PMV. The identities of pA and pB are less clear, although peptide mapping suggests that they and many of the minor products of translation are related to each other, but not to pC. The ability of partially encapsidated PMV ribonucleoprotein particles to direct protein synthesis has been assessed. As the extent of encapsidation increased, the relative synthesis of pA and pB decreased markedly. In contrast, the synthesis of pC persisted until a substantial fraction of the PMV RNA was completely encapsidated. In view of the polarity of assembly of PMV in vitro (M. Abouhaidar and J. B. Bancroft, (1978), Virology 90, 54-59) this finding indicates that the coding sequences for PMV coat protein are localized toward the 3' end of PMV RNA. A kinetic analysis of coat protein synthesis, coupled with Northern blot analysis of PMV RNA during translation suggest that PMV RNA must be cleaved to activate it for in vitro coat protein synthesis.     

In vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells.

Cytoplasmic RNA, isolated at various times after vaccinia virus infection, was translated in a message-dependent cell-free system prepared from rabbit reticulocytes. Supplementation of the system with calf liver tRNA specifically increased translation of viral RNA. Virtually all of the [³⁵S]methionine-labeled viral proteins from infected cells that were detected by sodium dodecyl sulfate -polyacrylamide gel electrophoresis appeared to be synthesized in the cell-free system. When programmed with RNA extracted at 2 hr after infection, early viral proteins were made and formation of cellular proteins was diminished. Primarily late proteins were synthesized using RNA extracted at 4 or more hr after infection, suggesting that the switch in protein synthesis is regulated principally by changes in RNA concentration rather than by modification of the translation apparatus of the cell. However, the vaccinia virus-mediated inhibition of host protein synthesis that occurred in the presence of actinomycin D was not associated with a decrease in translatable cellular mRNA. Immediate early RNA and early RNA were obtained by infecting cells in the presence of inhibitors of protein and of DNA synthesis, respectively. Analysis of the in vitro translation products did not reveal a class of early genes that require protein synthesis for expression. On the contrary, seven polypeptides, of which a 28,000-dalton species was most prominent, were synthesized in relatively greater amounts with immediate early RNA than with early RNA. All early and late mRNA species appear to be polyadenylylated, and a correlation between RNA sedimentation and molecular weight of translation product was obtained.     

Sodium dodecyl sulphate-polyacrylamide-gel electrophoretic detection of two high molecular weight proteins associated with tobacco mosaic virus infection in tobacco.

Proteins extracted from tobacco mosaic virus (TMV)-infected tobacco plants were analyzed by electrophoresis in sodium dodecyl sulphate (SDS)-polyacrylamide gels. A protein of ca. 150,000 daltons, not detectable in healthy plants, appeared in leaves infected for 2 days or more. A second, somewhat smaller protein (ca. 130,000 daltons) was found to accumulate in greater amounts; in the latter case, extracts of healthy plants also contained a protein migrating to the same position on the gels. The larger protein was detected in the 2500 and 20,000 g pellets of tissue homogenates fractionated by differential centrifugation, and the smaller one in the nonsedimentable (soluble) fraction.     

Nonstructural alfalfa mosaic virus RNA-coded proteins present in tobacco leaf tissue

The proteins synthesized under the direction of alfalfa mosaic virus RNAS in tobacco leaves have been examined under conditions of suppressed host protein synthesis. Besides the coat protein we could detect a 22K (K = apparent molecular weight in thousands), a 35K, and a set of 54K proteins. The 22K protein is serologically related to the coat protein. The 35K protein comigrated with the 35K protein whose synthesis is directed by RNA 3 in vitro The 54K proteins are serologically related to the 35K protein produced in vitro. Readthrough products of the 35K protein cistron into the coat protein cistron have been found previously in wheat germ extracts programmed with RNA 3. Two of these proteins comigrate with the 54K proteins synthesized in vivo. Since the 35K and the coat protein cistrons are read in different reading frames the formation of readthrough products is puzzling. In viruses with a tripartite genome the subgenomic mRNA for coat protein, RNA 4, is not known to be replicated as a separate genome entity. This might indicate that proteins synthesized by readthrough into the coat protein cistron play an essential role during replication, especially in the earliest phases.     

Structural polypeptides of adenovirus-associated virus top component.

Low density adenovirus-associated virus (AAV) from upper AAV-adenovirus bands on isopycnic CsCl gradients were purified by sedimentation through sucrose gradients and by immunoprecipitation of filtered particles. These particles appeared to be “empty” capsids because of their uptake of negative stain and their lack of isotopically labeled DNA. Analysis of these particles by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels demonstrated three polypeptides. These polypeptides showed identical molecular weights and the same relative concentrations as the structural polypeptides of marker complete AAV virions when compared by this technique. These findings indicate that the apparently “empty” AAV capsid does not lack any of the three polypeptides found in the complete virus particle, suggesting that none of them are core proteins.     

Electro-blot radioimmunoassay of virus-infected plant sap — A powerful new technique for detecting plant viruses

A new technique for detecting viruses in plant sap is described. It consists of sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the infected plant sap, electropheretic transfer of protein bands to activated paper by the Electro-Blot technique, the subsequent probing of the viral coat protein band by specific antiserum (prepared against intact virus), and detection of immune complex with ${}^{125}$I-labelled protein A. The technique successfully detected tobacco mosaic virus at a sap dilution of 1: 10,000, four strains of sugarcane mosaic virus (a potyvirus) in their perennial hosts infected for about 4 years, and five different isolates of potato leaf roll vkus (a luteovirus). The latter virus occurs in extremely low concentration and is difficult to detect by the other known methods.     

Translation of tobacco rattle virus RNA in vitro using wheat germ extracts.

When added to extracts from commercial wheat germ, tobacco rattle virus (TRV) RNA stimulated incorporation of radioactive amino acids into protein with an efficiency approaching that of tobacco mosaic virus (TMV) RNA. RNA from the smaller particle (RNA-2) of the CAM strain of TRV was translated largely into a single polypeptide which coelectrophoresed with coat protein and aggregated specifically with unlabeled protein. Coelectrophoresis with coat protein in 3% acrylamide gels indicated a C-terminal sequence in the radioactive product similar to that in coat protein. Attempts to change the pattern of translation products by heating RNA, adding coat protein to incubations, or changing RNA concentration were unsuccessful. RNA from the larger particle (RNA-1) of strain CAM (Campinas) was translated into a variety of products with a maximum molecular weight of 100,000. When mixtures of RNA-1 and RNA-2 were used, RNA-2 was translated preferentially.