Translational studies with turnip yellow mosaic virus RNAs isolated from major and minor virus particles

The major and minor nucleoprotein components in turnip yellow mosaic virus (TYMV) preparations have been fractionated by four cycles of CsCl gradient equilibrium centrifugations. RNAs isolated from the various fractions were translated in a rabbit reticulocyte cell-free system. Three classes of RNAs were studied: genomic RNA with an Mr of 2.0 x 10(6), the subgenomic coat protein messenger with an (r) of 2.4 x 10(5) (H. Guilley and J. P. Briand, 1978, Cell15, 113-122) and RNA molecules of intermediate sizes. Genomic RNA is found in the major TYMV components. These particles do not contain the coat protein messenger. The latter messenger has mainly been detected in two types of minor components, those which have an RNA content exceeding that of the major TYMV virions and particles which contain the coat protein messenger as their sole RNA constituent. RNAs of intermediate sizes are found in minor components less dense than the major particles; the full-length translational products of these RNAs and of genomic RNA overlap with one another and share a common NH2 terminus. It is concluded that a number of intermediate-sized RNAs and the genomic RNA have a common site for initiation of translation located close to their 5'-termini.     

Timing of the synthesis of empty shells and minor nucleoproteins in relation to turnip yellow mosaic virus synthesis in Brassica protoplasts

Following inoculation of Chinese cabbage protoplasts in vitro with turnip yellow mosaic virus, the development of characteristic changes in the chloroplasts was significantly asynchronous when observed by light or electron microscopy. In fully infected protoplasts there were only about 25% as many peripheral vesicles (the site of virus RNA synthesis) as in infected cells in the intact leaf. The yield of virus per infected protoplast was only about 1/10 th that found for an equivalent population of infected palisade cells from inoculated leaves. The production of empty protein shells was not reduced as much. Approximately equal amounts of virus nucleoprotein and empty protein shells were produced at all stages of the virus growth curve. However, if [35S]methionine was supplied when virus replication was well established, empty protein shells became labeled at an initial rate almost 10 times that of virus. Thus empty shells and virus must be assembled from differently located "pools" of protein subunits. The minor virus nucleoprotein fraction known to contain a substantial proportion of coat mRNA appears to be assembled earlier than the other minor nucleoprotein components, empty protein shells, or virus.     

Uncoating of turnip yellow mosaic virus RNA in vivo

Following mechanical inoculation of leaves with turnip yellow mosaic virus (TYMV), a significant proportion of the retained inoculum is uncoated within 45 sec, and the process is more or less complete after 2 min. At least 80-90% of the uncoating takes place in the epidermis. The application of virus to the intact leaf is essential for uncoating to occur. The uncoating process is not confined to plants which are known hosts for TYMV. The process gives rise to empty shells and low molecular weight protein. The empty shells probably lose a pentamer or hexamer of protein when the RNA is released. On a per cell basis the number of virus particles uncoated can be very large-approximately 106 particles per cell. The data suggest that at high inoculum concentrations most of the released RNA is inactivated on or within the epidermis.     

Properties of the DNA product of reverse transcription of turnip yellow mosaic virus RNA preparations.

Four populations of adenovirus type 16 incomplete particles with different buoyant densities in CsCl have been identified. They contain less DNA than complete particles. The standard viral genome has a molecular weight of 23 × 10⁶. The molecular weights of the incomplete DNA molecules range from 1 × 10⁶ to 16 × 10⁶, increasing with increasing density of the particles in CsCl. Characterization of isolated DNA by equilibrium density centrifugation in CsCl, DNA-DNA hybridization, and analysis of DNA labeled with [³H]thymidine before infection shows that both viral and host cell-specific DNAS are incorporated into incomplete virus particles.     

Infection of chlorophyll-less protoplasts from etiolated Chinese cabbage hypocotyls by turnip yellow mosaic virus

Protoplasts were prepared from hypocotyls of dark-grown, 8-day-old Chinese cabbage seedlings. Pectinase and cellulase were used together in a one-step procedure. These hypocotyl protoplasts did not contain detectable amounts of chlorophyll. They could be infected in vitro with turnip yellow mosaic virus (TYMV). Virus replication was demonstrated by direct immunofluorescence, enzyme-linked immunosorbent assay, virus recovery by sucrose density gradient centrifugation, and infectivity tests on chinese cabbage plants. The chlorophyll-less hypocotyl protoplasts supported TYMV replication in the dark as well as in the light, while chlorophyll-containing leaf protoplasts allowed TYMV multiplication only in the light.     

In vitro translation of adenovirus type 12-specific mRNA complementary to the transforming gene

Specific mRNA complementary to the adenovirus type 12 (AM) transforming gene was selected from Ad12-infected cells by hybridization with EcoRI-C (0–16.5 map units) and AccI-H (0–4.7 map units) DNA restriction fragments, and translated in vitro in [³⁵S]methio-nine-containing reticulocyte lysate. The products were analyzed by two-dimensional gel electrophoresis. The result showed that the EcoRI-C region encoded at least three polypeptides with molecular weights of 40,000 (40K), 38K, and 10K, while the AccI-H region encoded only 40K and 38K polypeptides. According to molecular weights and pI values, it is found that 40K or 38K and 10K polypeptides correspond to T antigen g and f, respectively. The result indicates that T antigen g is coded for by the left end of the transforming gene (0–4.7 map units, region la) and suggests that T antigen f is coded by the rest region (4.7–6.8 map units, region Ib). The size of mRNA for T antigen g was examined by translation with mRNA fractionated by sedimentation through a sucrose gradient and electrophoresis of ³²P-labeled mRNA selected by the Accl-H fragment. The results showed that the size of mRNA for T antigen g was about 13 S. The structure of the 13 S mRNA was examined by nuclease S1 mapping described by Berk and Sharp, and two :kinds of mRNA were detected. These two mRNAs are considered to be translated into two species of T antigen g polypeptides (40K and 38K).     

Identification of the human papovavirus T antigen and comparison with the simian virus 40 protein a.

The simian virus 40 (SV40) A protein (T antigen) and the early proteins produced by cells infected with BKV-type human papovaviruses have been compared by immunoprecipitation and tryptic peptide analyses. Cells infected with the human viruses and transformed cell lines synthesize phosphorylated proteins that interact with antiserum against the SV40 T antigen. These proteins cannot be distinguished from the SV40 A protein by sodium dodecyl sulfate-gel electrophoresis. Peptide map analyses have shown that the human virus proteins are similar, but not identical, to the SV40 A protein. Because of the distinct, reproducible differences in the peptides of the papovavirus proteins, structural analyses provide a direct procedure for detecting the expression of each type of virus in cells.     

Structural characterization of the Molluscum contagiosum virus genome

The genome of Molluscum contagiosum virus (MCV) appears in an electron microscope as a linear, duplex DNA molecule having a mean contour length of 53.02 ± 1.87 μm. Vaccinia virus DNA, similarly processed and used for comparison, measures 53.03 ± 2.2 μm. The average molecular weight of MCV-DNA was calculated to be 118 × 10⁶. Single-stranded circles measuring twice the length of linear molecules were observed with both poxvirus DNAs following high levels of denaturation. However, partial denaturation profiles of MCV and vaccinia virus DNA differed markedly when mounted for microscopy under similar conditions. MCV-DNA consistently required an additional 10° equivalents to achieve comparable levels of denaturation. In contrast to vaccinia virus DNA, the most easily denaturable regions of the MCV genome appeared to be the ends of the molecule. When MCV-DNA was cleaved by restriction endonuclease BAM · HI, 12 fragments were produced ranging in size from 4 × 10⁵ to 31 × 10⁶ daltons as determined by agarose-gel electrophoresis and contour length measurements. Vaccinia virus DNA, similarly treated, produced more than 23 fragments which did not appear to correspond in size to those of MCV. With the aim of comparing the genetic and structural heterogeneity of the MCV genome, 11 independently isolated virus samples were analyzed by restriction endonuclease digestion and coelectrophoresis. Only three cleavage profiles were detected, with some fragments present in all three profiles, comigrating in agarose gels. We conclude, therefore, that the genome of Molluscum contagiosum virus is a continuous polynucleotide chain which is base paired to form a linear, duplex DNA molecule. Three genetic types of MCV were isolated from clinically typical skin lesions and identified in this study.     

Activation of Prunus necrotic ringspot virus and rose mosaic virus by RNa 4 components of some llarviruses

RNA extracted from rose mosaic virus (RMV) and from necrotic ringspot virus-G(NRSV) separated into four sedimenting components in sucrose density gradients. The components were designated RNAs 1, 2, 3, and 4 in order of decreasing sedimentation velocities. Sodium dodecyl sulfate (SDS)-denatured NRSV and RMV preparations showed a prominent protein species of about 25,000 daltons when electrophoresed in polyacrylamide gels. In addition, two closely migrating protein species of about 19,000 daltons were also observed in SDS-denatured RMV preparations. Preparations containing RNA 1+2+3 and preparations containing RNA 4 were obtained by two successive cycles of sucrose density gradient centrifugation. Neither preparation was infectious alone. Mixtures of RNA 1+2+3 from each virus became infectious, however, with the addition of homologous RNA 4. RMV protein also activated RMV-RNA 1+2+3 preparations. NRSV-RNA 4 efficiently activated RMV-RNA 1+2+3, but RMV-RNA 4 had very little ability to activate NRSV-RNA 1+2+3 even though it efficiently activated its homologous RNA 1+2+3 preparations. RNA 4 preparations from alfalfa mosaic virus (AMV) and citrus leaf rugose virus (CLRV) activated RMV-RNA 1+2+3. Comparable concentrations of RMV-RNA 4, however, failed to activate RNA 1+2+3 from AMV and from CLRV. RNA from CLRV, RMV, and citrus variegation virus (CVV) efficiently uncoated AMV particles. However, AMV-RNA and CLRV-RNA 4 uncoated CVV but not CLRV or RMV. The evidence presented indicated that viruses in the new Ilarvirus group [Shepherd, R. J., et al. (1976). Intervirology 6, 181–1841 have similar infectivity requirements; that is, mixtures of RNA 1+2+3 are not infectious but can be activated by either RNA 4 or coat protein.     

The formation of fast-sedimenting turnip yellow mosaic virus RNA: structural rearrangement inside the capsid.

Treatment of TYMV (turnip yellow mosaic virus) virions under alkaline conditions and at high ionic strength leads to the in situ formation of RNA sedimenting uniformly at 38 S. Native TYMV-RNA has a sedimentation coefficient of 28 S in the same solvent. A systematic study in which the temperature, ionic strength, and pH were varied showed that favorable conditions for 38 S RNA formation are 1.0 M KCl, pH 10.5, 30°, and a reaction time of 5–10 min. Nonetheless, the fast-sedimenting RNA can also be obtained at neutral pH and 1.0 M KCl, provided that the time of treatment is prolonged or the temperature is raised. No evidence for breakage of the RNA chain was obtained under the latter conditions, while all of the RNA is retained by the virion. These data indicate that exposure of TYMV to high ionic strength causes a drastic structural rearrangement of the viral RNA inside the capsid without concurrent fragmentation of the RNA chain.     

In vitro mutagenesis of a putative DNA binding domain of SV40 large-T

A large number of deletion and point mutations were introduced into a small region of the SV40 large-T gene that was believed to encode part of a DNA-binding domain. The majority of mutant proteins constructed were unable to stimulate viral DNA replication, but all retained at least some transforming activity. Those replication-defective mutants with lesions affecting amino acid residues between 144 and 156 were postulated also to be defective in the sutoregulation function of large-T to account for their ability to transform Rat-1 cells more avidly than wild-type. Two mutants (Glu 107 → Lys and Ser 189 → Asn) were isolated which exhibited severely reduced transforming activity but which supported normal rates of virus and viral DNA replication. Mutation of individual serine and threonine phosphorylation sites within the amino-terminal half of large-T had little effect on the protein's transforming activity. These and other mutations that affected amino acid residues either side of the region from 127 to 133, previously shown to be essential to the nuclear localisation of large-T [D. Kalderon, W. D. Richardson, A. F. Markham, and A. E. Smith (1984) Nature (London)311, 33–38] did not discernibly impair nuclear accumulation.     

The incorporation of radiolabeled polyamines and methionine into turnip yellow mosaic virus in protoplasts from infected plants

Turnip yellow mosaic virus contains large amounts of nonexchangeable spermidine and induces an accumulation of spermidine in infected Chinese cabbage. By 7 days after inoculation, a majority of protoplasts isolated from newly emerging leaves stain with fluorescent antibody to the virus. These protoplasts contain 1-2 X 10(6) virions per cell and continue to produce virus in culture for at least 48 hr. [14C]Spermidine (10 microM) was taken up by these cells in amounts comparable to the original endogenous pool within 24 hr. However, after an initial rise, the spermidine content of the cell returned to its original level, implying considerable regulation of the endogenous pool(s). Putrescine and spermine were major products of the metabolism of exogenous spermidine. Radioactivity from exogenous [14C]spermidine was also readily incorporated into the ribonucleoprotein component(s) of the virus, where it appeared as both spermidine and spermine. The specific radioactivities of the viral polyamines were approximately twice those of spermidine and spermine extracted from the whole cell. Radioactivity from [2-14C]methionine was readily incorporated into the protein, spermidine, and spermine of the virus. Again, the specific activities of these amines were substantially higher in the virus than in the whole cell. Thus, newly formed virus contained predominantly newly synthesized spermidine and spermine. However, inhibition of spermidine synthesis by dicyclohexylamine led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virus.     

Size and complexity of polyadenylated RNAs induced in tobacco infected with sonchus yellow net virus

Hybridization of 32P-labeled sonchus yellow net virus (SYNV) RNA to polyadenylated (poly A+) RNA from infected tobacco reveals the presence of four electrophoretically distinct components. These components probably represent five discrete RNA species complementary to SYNV RNA (scRNAs). The scRNAs are smaller than the 13,000 nucleotide (NT) SYNV genome and range in size from 1200 to 6600 NT. Individual recombinant DNA clones derived from SYNV RNA hybridize to at least three and probably four of the scRNAs. These results suggest that each of the scRNAs contains unique sequences with a combined size representing more than 90% of the viral genome. Therefore, the size range and sequence complexity of the scRNAs are as expected for messenger RNAs encoding the four major SYNV polypeptides and the minor "L-protein."     

In vitro and in vivo translation of the ribonucleic acids of a cowpea strain of tobacco mosaic virus1

Experiments reported here support the notion that intact TMV RNA has genes which are not translated directly and that specific fragments of the RNA are the functional messengers for those genes.A discrete class of short nucleoprotein rods, in addition to the expected 300-nm rods, was observed by Morris in a cowpea strain of TMV isolated from infected cowpea tissue. We have found a third class of rods of intermediate length. RNAs from the three kinds of rods were separately translated in a wheat germ cell-free protein synthesis system. RNA from the short rods directed the synthesis of a polypeptide with some properties of capsid protein: the same electrophoretic mobility in detergent-impregnated polyacrylamide gels and the ability to participate in a reconstitution reaction with virus RNA. RNA from long rods directed the synthesis of a spectrum of polypeptides. Among these one of ～130,000 molecular weight predominated. It had the same electrophoretic mobility as a polypeptide which was recovered from virus-infected tissue, but not from mock-inoculated tissue. RNA from rods of an intermediate length class directed the synthesis of one or two polypeptides with apparent molecular weights of about 30,000. Capsid polypeptide could not be demonstrated among the products formed under the direction of the long or immediate rods RNAs. However, RNA-RNA hybridization experiments indicate that short and intermediate rod RNAs share nucleotide sequences with each other and with the long rod RNA. The possible relationships of these results to the replication of the common strain of TMV and brome mosaic virus are discussed.     

A comparative study on the in vitro translation products of individual RNAs from two-, three-, and four-component strains of barley stripe mosaic virus

Passaging through plants of the three-component barley stripe mosaic virus (BSMV) strain Norwich (Norwich III) yielded a two-component isolate (Norwich II). A study was made of the sets of polypeptides translated in a rabbit reticulocyte and wheat embryo cell-free systems from individual RNAs of (1) the natural three-component strain Norwich III, (2) a two-component isolate derived from the former (Norwich II), and (3) an isolate intermediate between the three- and two-component ones, Norwich;. Translation of RNA 1 from all three variants of the Norwich strain in vitro yields a single major product with a molecular weight (Mr) of 120,000 (p120). RNA 2 from Norwich III in vitro produces two polypeptides: the viral coat protein (p23) and, in certain ionic conditions, a polypeptide of 25,000 M(r) (p25). RNA 3 from Norwich III is translated into a protein with Mr of 75,000 (p75). Conversion of Norwich III into Norwich II is accompanied by the changes in the coding specificity of RNA 2; beside the coat protein and p25, a protein of 85,000 M(r) (p85) is formed upon its translation-a feature characteristic of RNA 2 of the naturally occurring bipartite BSMV strains (Russian, type). With the Norwich(i) variety, which is marked by a significantly reduced portion of RNA 3 in the total virion RNA preparation, RNA 2 in addition to the coat protein, p25, and p85 directs the synthesis of another product with M(r) of about 78,000. Thus, in conversion of the three-component BSMV into a two-component one, the loss of RNA 3 is concomitant with the actuation in RNA 2 of a sequence coding for p85. RNAs 1-3 of the quadripartite Argentina Mild (AM) BSMV strain code in vitro for the same polypeptides as RNAs 1-3 of Norwich III. AM RNA 4 is translated as a monocistronic template into a polypeptide with Mr of 55,000 (p55). Amino acid sequences of p85, p75, and p55 are shown to overlap among these polypeptides but not to appreciably overlap with p120 sequences. Data presented here allowed a tentative structure to be proposed for genome of two-, three-, and four-component BSMV strains.     

Some properties of carnation ringspot virus single- and double-stranded ribonucleic acid

From sodium dodecyl sulfate (SDS)-dissociated carnation ringspot virus (CRSV) two RNAs of different sizes were separated by density gradient centrifugation. Preparations of the smaller RNA-2 (0.5 × 10⁶ daltons) were not infectious; preparations of the larger RNA-1 (1.5 × 10⁶ daltons) were only moderately infectious. Mixtures of the two kinds of preparations were very infective. This enhancement of infectivity was demonstrated within and between the two RNAs from each of two strains of CRSV. The genetic information controlling irreversible virus particle aggregation and dissociation of virus particles by SDS at pH 5 was contained in RNA-1. Virus-infected tissue contained dsRNA replicative forms corresponding to RNA-1, RNA-2, and a minor ss-RNA component.     

Properties of the nucleocapsids of a virus isolated from Oryctes rhinoceros.

Nucleocapcids were isolated from purified particles of “Oryctes” virus (a candidate Baculovirus) by treatment with Nonidet P-40. The nucleocapsids were homogeneous in size and density and contained the viral DNA as well as eight of the twelve virus particle polypeptides resolved on polyacrylamide gels. The nucleocapcids were serologically related to nucleocapcids of another Baculovirus (a nuclear polyhedrosis virus from Spodoptera littoralis), although the “Oryctes” nucleocapcids were morphologically distinct in carrying a novel tail-like projection at one end of the particle.     

The generation of the two envelope glycoproteins of Rous sarcoma virus from a common precursor polypeptide.

The double-stranded genome RNAs of recombinants between reovirus serotypes 1, 2, and 3 were examined by polyacrylamide-gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed construction of a map of the serotypes correlating genome segments providing functions interchangeable between the serotypes. The relative migration rates of segments M1 and M2 of type 3 are reversed between the traditional Tris-acetate-buffered gel system and the Tris-glycine gel system used here. In the Tris-glycine system, the genome segments of serotype 1 correspond to type 3 in order of increasing electrophoretic mobility except for S3 and S4 which are reversed. In serotype 2 all segments except M1 and M2 and S3 and S4 correspond in order of increasing electrophoretic mobility. The migration of these two segment pairs is reversed in type 2 relative to type 3. A map is presented correlating the migration of genome segments of types 1, 2, and 3 in both the Tris-glycine- and the Tris-acetate-buffered systems. The nomenclature of the genome segments is standardized to that which appears in the literature. In addition, these data demonstrate that recombinants arise by physical reassortment of genome segments between parents.     

The effects of dicyclohexylamine on polyamine biosynthesis and incorporation into turnip yellow mosaic virus in Chinese cabbage protoplasts infected in vitro

We have reported (R. Balint and S. S. Cohen, 1985, Virology 144, 181-193) that protoplasts from plants infected with turnip yellow mosaic virus (TYMV) continue to produce virus in culture and that newly formed virus particles contained predominantly newly synthesized spermidine and spermine. Inhibition of spermidine synthesis by dicyclohexylamine (DCHA), however, led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virions. We now report similar results with healthy protoplasts infected in vitro, in which essentially all of the virus is newly formed. Again, newly synthesized spermidine and spermine were preferentially incorporated into virus. DCHA inhibited spermidine synthesis by 85%, leading in 20 hr to a 60% depletion of the cellular spermidine and a 30% reduction in the amount of spermidine per virion. Spermine synthesis increased, however, producing a 40% increase in cellular spermine and 50-100% increase in the amount of spermine per virion. Thus, in spite of spermidine depletion, the total positive charge contributed by polyamines to the virus was essentially conserved.