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Flockhouse virus (FHV) is a member of the nodavirus group of positive-strand RNA viruses. In the absence of additional compounds, a template-dependent RNA-dependent RNA polymerase extracted from FHV-infected cells synthesizes complementary (-)-strand copies of added FHV RNA to yield a double-stranded RNA product. Upon addition of glycerophospholipid (GPL), this system reproducibly carries out complete highly active replication of added FHV RNA, producing newly synthesized (+)-strand RNA in predominantly single-stranded RNA form. This accounts for previously observed effects of Lipofectin (a mixture of GPL and cationic lipid) in the system. All tested neutral and negatively charged GPLs except phosphatidic acid support complete FHV RNA replication in this in vitro system, as do phospholipid extracts from uninfected and FHV-infected cells. Neither sphingomyelin, a membrane phospholipid that is not derived from glycerol, nor cholesterol supported FHV RNA replication. Testing of compounds derived from GPL shows that the ability of active GPL to support FHV (+)-strand RNA synthesis is dependent on the structures of both the head group and the acyl chains. Neither the phosphorylated head group nor the diacylglycerol lipid moiety alone supports RNA replication. The length and saturation of acyl chains strongly influence the ability of GPL to support RNA replication. Other characteristics of this in vitro RNA replication system and the possible role played by membranes and their components in FHV RNA replication are discussed.

Newly synthesized virions of flock house virus (FHV), an insect nodavirus, were detected in plant cells inoculated with FHV RNA. FHV was found in whole plants of barley (Hordeum vulgare), cowpea (Vigna sinensis), chenopodium (Chenopodium hybridum), tobacco (Nicotiana tabacum), and Nicotiana benthamiana and in protoplasts derived from barley leaves. Virions produced in plants contained newly synthesized RNA as well as newly synthesized capsid protein. These results show that the intracellular environment in these plants is suitable for synthesis of a virus normally indigenous only to insects. Such synthesis involves, minimally, translation of viral RNA, RNA replication, and virion assembly. Inoculation of barley protoplasts with FHV virions resulted in synthesis of small amounts of progeny virions, suggesting that FHV virions are capable of releasing their RNA in plant cells. In N. benthamiana, virions resulting from inoculation with RNA were detected not only in inoculated leaves but also in other leaves of inoculated plants, suggesting that virions could move in this plant species. Such movement probably occurs by a passive transport through the vascular system rather than by an active transport involving mechanisms that have evolved for plant viruses.

Cell-free extracts of Drosophila melanogaster cells infected with FHV (Flockhouse virus) have the capacity to synthesize FHV RNA. When such extracts are incubated appropriately, ss and ds FHV RNA 1, -2, and -3 can be detected as synthesis products. Pulse-chase analyses indicate that radioactivity, incorporated into ds RNA 1, -2, and -3 is chased into ss RNA 1, -2, and -3, suggesting that these ds RNAs are intermediates in FHV RNA replication. When the cell-free extracts are treated with detergents to solubilize cellular membranes, only ds RNA 1, -2, and -3 can be detected as synthesis products. The cell-free extracts, treated with a ribonuclease to remove endogenous templates, have the capacity to replicate FHV RNA 1 and -2 when these RNAs are provided as templates, together with lipofectin. Both as and ds RNA 1 and -2 are detected as synthesis products. When RNA3 is provided as a template, only ds RNA3 is synthesized. In the absence of lipofectin or in the presence of detergent, only ds RNAs are detected as synthesis products and all the incorporated radioactivity is found in the strand complementary to that of the template. Our results are consistent with a mechanism in which second-strand synthesis, although not complementary strand synthesis, requires intact membranes. They also suggest that ds RNA3 is an intermediate in the synthesis of ss RNA3.

The genomic RNA2s of nodaviruses encode a single gene, that of protein alpha, the precursor of virion proteins beta and gamma. We compared the sequences of the RNA2s of the nodaviruses, black beetle virus (BBV), flock house virus, boolarra virus and nodamura virus, with the objective of identifying homologies in the primary and secondary structure of these RNAs and in the structure of their encoded protein. The sequences of the four RNAs were found to be similar, so that homologous regions relating to translation and RNA replication were readily identified. However, the overall, secondary structures in solution, deduced from calculations of optimal Watson-Crick base-pairing configurations, were very different for the four RNAs. We conclude that a particular, overall, secondary structure in solution within host cells is not required for virus viability. The partially refined X-ray structure of BBV (R = 26.4% for the current model) was used as a framework for comparing the structure of the encoded proteins of the four viruses. Mapping of the four protein sequences onto the BBV capsid showed many amino acid differences on the outer surface, indicating that the exteriors of the four virions are substantially different. Mapping in the beta-barrel region showed an intermediate level of differences, indicating that some freedom in choice of amino acid residues is possible there although the basic framework of the capsids is evidently conserved. Mapping onto the interior surface of the BBV capsid showed a high degree of conservation of amino acid residues, particularly near the protein cleavage site, implying that that region is nearly identical in all four virions and has an essential role in virion maturation, and also suggests that all four capsid interior surfaces have similar surfaces exposed to the viral RNA. Apart from a small portion of the C promoter, the amino terminus of the BBV protein (residues 1 to 60) is crystallographically disordered and the amino acid residues in that region are not well conserved. The disordered portion of the BBV protein clearly projects from the capsid inner surface into the interior of the virion, the region occupied by the viral RNA. In all four viruses, residues 1 to 60 had a high proportion of basic residues, suggesting a virus-specific interaction of the amino terminus with the virion RNA.

We have adapted the chain-termination method for determining the nucleotide sequence of DNA of Sanger, Nicklen, and Coulson [(1977) Proc. Natl. Acad. Sci. USA 74, 5463--5467] for use with reverse transcriptase (RNA-directed DNA nucleotidyltransferase) on RNA templates. With this method and using a primer (the octanucleotide pdT7rC) directed at the 3'-terminal poly(A), we have determined a sequence of 166 residues in the genomic RNA of the picornavirus encephalomyocarditis virus.

Turnip crinkle virions contain genomic RNA, Mr approximately 1.4 x 10(6), small RNA, Mr approximately 1.5 x 10(5) and a heterogeneous collection of intermediate size. Turnip crinkle virion RNA was translated in rabbit reticulocyte lysates into three major unrelated products of molecular weight 38,000 (p38, the virion coat protein), 36,000 (p36), and 25,000 (p25). The same three products, but in different proportion, were synthesized in wheat embryo extracts. Certain intermediate size RNA fractions translated into p38 and p36 products much more efficiently than did unfractionated RNA. The 1.5 x 10(5) molecular weight RNA did not induce amino acid incorporation. We propose a translation map of the TCV genome, based on tryptic analyses of the major products and of putative read-through proteins.

RNA isolated from southern bean mosaic virions contains, in small amount, a subgenomic RNA (molecular weight, 0.38 × 10 6 ) that serves in vitro as an mRNA for southern bean mosaic virus coat protein. The RNA has a 5′-linked protein indistinguishable from the protein linked to the 5′ end of full-length genomic RNA. Its base sequence, determined to 91 bases from the 3′ end, is identical to the 3′-terminal sequence of the genomic RNA. The results suggest that the coat protein messenger sequence exists as a “silent” cistron near the 3′ end of the genomic RNA.

Genetic recombination of DNA is one of the fundamental mechanisms underlying the evolution of DNA-based organisms and results in their diversity and adaptability. The importance of the role of recombination is far less evident for the RNA-based genomes that occur in most plant viruses and in many animal viruses. RNA recombination has been shown to promote the evolutionary variation of picornaviruses1–4, it is involved in the creation of defective interfering (DI) RNAs of positive- and negative-strand viruses5–9 and is implicated in the synthesis of the messenger RNAs of influenza virus10 and coronavirus11. However, RNA recombination has not been found to date in viruses that infect plants. In fact, the lack of DI RNAs and the inability to demonstrate recombination in mixedly infected plants has been regarded as evidence that plants do not support recombination of viral RNAs. Here we provide the first molecular evidence for recombination of plant viral RNA. For brome mosaic virus (BMV), a plus-stranded, tripartite-genome virus of monocots, we show that a deletion in the 3′ end region of a single BMV RNA genomic component can be repaired during the development of infection by recombination with the homologous region of either of the two remaining wild-type BMV RNA components. This result clearly shows that plant viruses have available powerful recombinatory mechanisms that previously were thought to exist only in animal hosts, thus they are able to adapt and diversify in a manner comparable to animal viruses. Moreover, our observation suggests an increased versatility of viruses for use as vectors in introducing new genes into plants.

The genomic RNA sequence of the cowpea strain of southern bean mosaic virus (SBMV-C) has been determined. The genome is 4194 nucleotides in length and has four open reading frames. A 5' proximal open frame, from base 49 to base 603, corresponds to the length of the P4 proteins translated in cell-free extracts from full-length and smaller virion RNA. The largest open frame extends from base 570 to base 3437 and encodes the two largest proteins translated in cell-free extracts from full-length virion RNA. Segments of this open reading frame's predicted amino acid sequence resemble those of known viral RNA polymerases, ATP-binding proteins, and viral genome-linked proteins. A third open frame extends from base 1895 to base 2380 and has not been correlated with an in vitro translation product. The fourth open reading frame is located in the 3' terminal region of the genome extending from base 3217 to base 4053. This frame encodes the SBMV capsid protein which is translated from subgenomic, virion RNA.

The black beetle virus (BBV) is an isometric insect virus whose genome consists of two messenger-active RNA molecules encapsidated in a single virion. The nucleotide sequence of BBV RNA1 (3105 bases) has been determined, and this, together with the sequence of BBV RNA2 (1399 bases) provides the complete primary structure of the BBV genome. The RNA1 sequence encompasses a 5' non-coding region of 38 nucleotides, a coding region for a protein of predicted molecular weight 101,873 (protein A, implicated in viral RNA synthesis) and a 3' proximal region encoding RNA3 (389 bases), a subgenomic messenger RNA made in infected cells but not encapsidated into virions. The RNA3 sequence starts 16 bases inside the coding region of protein A and contains two overlapping open reading frames for proteins of molecular weight 10,760 and 11,633, one of which is believed to be protein B, made in BBV-infected cells. A limited homology exists between the sequences of RNA1 and RNA2. Sequence regions have been identified that provide energetically favorable bonding between RNA2 and RNA1 possibly to facilitate their common encapsidation, and between RNA2 and negative strand RNA1 possibly to regulate the production of RNA3.

RNA transcripts of cloned cDNA of the genomic RNAs of BBV (black beetle virus) are infectious to cultured cells of Drosophila melanogaster. Individual transcripts had approximately 10% of the infectivity of the corresponding authentic virion RNA. Progeny virus resulting from transcript infection was phenotypically indistinguishable from the progenitor virus used to generate the original cDNA forms as judged by sucrose density gradient sedimentation, specific infectivity, plaque morphology, and serology. Although the transcript RNAs used to produce this virus had 20 nonviral bases headed by a capping group at their 5' termini, these 20 bases were absent in the progeny viral RNAs. The cDNA forms, and therefore the resulting transcript RNAs, should be readily modifiable by the techniques of recombinant DNA technology both for viral studies and for the insertion of foreign genes into the viral genome and thus into the host cytoplasm.

Rapid, detailed, and accurate analysis of the length spectrum of 3' terminal poly(A) in an RNA population can be obtained by 3'-terminal 32P-labelling of RNA with T4 RNA ligase, digestion with ribonucleases T1 and A, and use of gel sequencing methods. Length distributions of 3'-terminal poly(A) of EMC virus, poliovirus, rhinovirus, RAV-61, and CPMV virion RNAs as well as mouse globin mRNA are presented.

All four components of brome mosaic virus RNA have m 7 G 5′ ppp 5′ Gp as their 5′ terminus. The m 7 G can be removed by β-elimination, resulting in the conversion to pppGp.

Nuclease digestion of SBMV RNA releases a protein of molecular weight approximately 12,000 in addition to the four mononucleotides. The lack of reactivity of SBMV RNA to polynucleotide kinase and the absence of a capping group suggest that the protein is covalently attached to the 5' end of the RNA. RNA sequencing shows that the 3' terminus of SBMV RNA is not polyadenylated.

The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.

Southern bean mosaic virus RNA was translated in wheat embryo extracts and in nuclease-treated rabbit reticulocyte lysates. Four principal products were synthesized: two related products with molecular weights of 105,000 and 75,000, a product with a molecular weight of 29,000 that closely resembled coat protein, and a product with a molecular weight of 14,000. Their proportion depended on ionic conditions and the translational system used.

A 3'-terminal oligonucleotide fragment, 161 bases long, can be obtained from each of the four brome mosaic virus RNAs by means of nuclease digestion. Like the four intact brome mosaic virus RNAs, each fragment accepts tyrosine in a reaction catalyzed by wheat germ aminoacyl-tRNA synthetase. The complete nucleotide sequence of the RNA 4 fragment has been determined by use of standard radiochemical methods. Comparative data for the fragments from RNAs 1, 2, and 3 show that they have nearly the same sequence as the RNA 4 fragment. The eight bases adjacent to the 3' terminus of the RNA 4 fragment are identical in sequence to the eight terminal bases of tyrosine tRNA from Torula utilis and eleven interior bases are identical in sequence to eleven bases encompassing the anticodon region of tyrosine tRNA from Saccharomyces cerevisiae, T. utilis, and Escherichia coli. Nevertheless, reasonable base-pairing schemes yield, at best, a distorted cloverleaf secondary structure.

The RNA from bacteriophage Qβ can be translated by cell-free extracts from wheat embryos. This translation, by 80 S ribosomes, occurs at a low magnesium ion concentration. Three products are synthesized which coelectrophorese with Qβ proteins synthesized in Escherichia coli extracts. The smallest of these has been identified as coat protein. Although the polycistronic bacteriophage message is translated with fidelity, the efficiency is much less than when the monocistronic brome mosaic virus coat protein message is translated.

A cell-free protein synthesizing system was prepared from cells of Drosophila melanogaster line 1 and made mRNA dependent by treatment with micrococcal nuclease. The system was tested with homologous RNA from black beetle virus propagated in Drosophila cells, with Drosophila heat shock mRNA, and with various heterologous viral mRNA's. Under optimal conditions amino acid incorporation programmed with black beetle virus RNAs was 30-fold higher than endogenous incorporation. RNAs 1 and 2 primarily directed the synthesis of proteins with approximately molecular weights of 120,000 and 46,000, respectively. mRNA's, prepared by transcription from vesicular stomatitis virus or vaccinia virus, were translated efficiently and yielded products that comigrated with authentic viral proteins. Brome mosaic virus RNA and encephalomyocarditis virus RNA were translated poorly. The system retained full activity after freezing.

Crude lysates of black beetle virus (BBV)-infected cells of Drosophila melanogaster contain an RNA-dependent RNA polymerase activity not detectable in uninfected cells. The activity (designated BBV polymerase) sedimented at 20,000 × g , indicating an association with particulate material. It was solubilized from the pellet by sonication in a magnesium-deficient buffer. Differential centrifugation resulted in a 43-fold purification with 84% recovery of polymerase activity. The effects of divalent and monovalent cations, time, temperature, and pH on the activity of the partially purified polymerase were examined. RNA synthesis was not stimulated by the addition of exogenous BBV RNA, suggesting that an enzyme-template complex existed. Analysis of the RNA products of the RNA polymerase reaction indicated that full-length “negative” strand BBV RNAs were synthesized.

3' terminal fragments of BMV RNA as short as 153 bases in length serve as efficient templates in vitro for BMV-specific RNA polymerase. Template activity of such fragments or of native BMV RNA is abolished when cDNA fragments as short as 39 bases are hybridized to their 3' termini. Hybridization of cDNa fragments to regions of BMV RNA 200 or more bases distal to the 3' end has no discernible effect on initiation and little effect on elongation. We conclude that BMV RNA polymerase initiates binding with an RNA template through a mechanism mediated by the tRNA-like 3' end of BMV RNA, requiring at least some of the last 39, but no more than the last 153 bases.

When RNA from southern bean mosaic virus is fractionated on a sucrose gradient, the resulting absorbance profile shows a major peak corresponding to 1.4 × 10 6 MW together with a considerable amount of slower sedimenting material. The RNA from these gradient fractions was translated in wheat embryo extracts and reticulocyte lysates. Only RNA in the molecular weight range 0.3 – 0.4 × 10 6 was found to induce synthesis of coat protein (designated P3). RNA of molecular weight 1.4 × 10 6 induced synthesis of three proteins, P1, P2, and P4.

The 3' terminal 337 bases of BMV (brome mosaic virus) coat protein mRNA (BMV RNA4) are presented. This sequence includes the terminal portion of the coat protein cistron and the complete 300-base 3' noncoding sequence. The 3' noncoding sequence displays significant complementarity to the 5' terminal sequence of BMV RNA3 but not to the 5' terminal sequence of BMV RNA4.

The nucleotide sequence of black beetle virion (BBV) RNA2 has been determined. RNA2 is 1399 b long. Its 5' terminus is capped. Its 3' terminus has an unidentified moiety that renders the RNA resistent to polyadenylation and ligation. The first AUG codon at base 23 is followed by an open reading frame for a protein 407 amino acids long, the predicted size of coat protein precursor. A second open reading frame for a putative protein 72 amino acid residues long begins at base 1110. No other large open reading frames exist. The 5' half of the RNA can be folded into a long, imperfect hairpin of high predicted stability. The 3' half of the RNA can fold into a complex set of multiply bifurcated stem and loop regions.

Unfractionated bromegrass mosaic virus RNA and each of its three naturally occurring components, of molecular weight 1.0 × 10 6 , 0.7 × 10 6 , and 0.3 × 10 6 , stimulate the incorporation of amino acids into peptides in an E. coli cell-free incorporating system. Analysis of tryptic digests by paper electrophoresis and chromatography indicated that the products induced by the RNA small component of molecular weight 0.3 × 10 6 , and by the unfractionated RNA were similar.

The RNA's from the bacteriophages M12 and R17, were digested with pancreatic ribonuclease to yield pyrimidine mononucleotides and pyrimidineterminated oligonucleotides. All the non-isomeric digestion products were separated by ion exchange chromatography. The complete set of such fragments has been designated the pyrimidine catalog of the RNA. M12 RNA and R17 RNA were found to have very similar pyrimidine catalogs and, by inference, very similar RNA sequences.

X-ray scattering data are presented on solutions of wild cucumber mosaic virus and the associated “top component” particles which have little or no RNA. The radii of gyration are 112 Å and 135 Å for bottom and top component, respectively. The radial density distribution within each particle is calculated by Fourier inversion of the scattered amplitudes. The virus particle or bottom component has approximately uniform density with an outer radius of about 140 Å. The transform of the top component shows an almost hollow center extending out to 105 Å with a surrounding shell of high density about 35 Å thick. Thus the RNA would appear to occupy the region inside 105 Å and does not overlap appreciably the region occupied by protein. The virus has associated with it approximately 0.38 gm of water per gm of virus, resulting in an average electron density of 1.25 times that of water.

Most of the large oligonucleotides arising from pancreatic ribonuclease digestion of R17 and M12 RNA's have been isolated and sequenced. The majority of the R17 and M12 RNA sequences are identical or similar and some are also identical with reported MS2 RNA sequences. The data strongly imply that these three RNA's are almost identical.

The infectivity of bromegrass mosaic virus (BMV) RNA has been investigated. The infectivity per unit weight of unfractionated RNA was approximately one-third that of an equivalent weight of RNA in the virus. No significant loss of infectivity occurred during 70 hours of incubation at 30°. BMV RNA is known to consist of three components of molecular weights 0.3 × 10 6 , 0.7 × 10 6 , and 1.0 × 10 6 . These components were separated by sucrose density-gradient centrifugation. It was found that infectivity was associated only with the 1.0 × 10 6 molecular weight component; the two smaller components were noninfectious when tested either separately or in combination. An enhancement of infectivity was observed when the infectious component was inoculated in the presence of either of the smaller components or in the presence of unrelated RNAs.

The protein and RNA of bromegrass mosaic virus were separated from each other by dialysis of the virus against 1 M -calcium chloride. In this medium the virus is degraded, the RNA is quantitatively precipitated and there remains a supernatant solution of viral protein. The protein obtained in this way was soluble in various buffers in the pH range 5·5 to 6·5 and exhibited a single symmetrical sedimenting boundary in the ultracentrifuge. Its molecular weight was 40,000 ± 2,000 as determined by the Ehrenberg method of approach to sedimentation equilibrium. Amino acid analyses indicated that there were about 52 lysyl plus arginyl residues per molecular weight of 40,000. Tryptic hydrolysis of urea-denatured protein yielded 23 soluble peptides and an insoluble residue amounting to about 20% by weight of the intact protein. From the number of soluble tryptic peptides and an assumed number of tryptic peptides in the insoluble residue it was concluded that the molecular weight of the protein subunit of bromegrass mosaic virus was slightly greater than 20,000 and that the protein moiety obtained by treatment with 1 M -CaCl 2 was a relatively stable dimer of the subunit. Partial separation of the soluble peptides resulting from tryptic hydrolysis was achieved on a preparative scale by column chromatography.

The drug rifampin, when added at the time of infection, inhibits synthesis of the phage Qβ. Both viral ribonucleic acids and viral proteins are made in nearly the same amount as in the absence of rifampin, but the rate of assembly into phage particles is low.

Broad bean mottle virus can be degraded by dialysis against 1 M -CaCl 2 . The virus RNA is quantitatively precipitated together with some of the protein. Most of the virus protein, free of RNA, can be recovered from the supernatant liquid. The amino acid composition of the protein was determined. End group analyses were performed on the oxidized protein. The C-terminal amino acid was found to be alanine. No N-terminal amino group was found. The tryptic peptides of the oxidized protein were separated by a two-dimensional combination of paper chromatography and electrophoresis. Twenty-eight peptides were detected. From the number of tryptic peptides and the amino acid composition, the molecular weight of the protein subunit was calculated to be 20,500.

When subjected to electrophoresis in polyacrylamide gels, the virions of wild-type Qbeta bacteriophage are found in a single, major, anomalously wide band. With Qbeta mutant 27-2, this wide band is replaced by a set of narrow, well-defined bands. The most rapidly migrating band of the mutant, comprising less than 10% of the total, contains defective virions. These virions have sedimentation coefficients ranging from 70 to 100% of the bulk of the unfractionated mutant, they contain no read-through protein (protein IIb), and they are deficient in maturation protein and contain fragmented RNA. The second band, comprising less than 3% of the total virus, has not been well characterized. The virions in the remaining electrophoretic bands are infective. Their distribution into bands is believed due to differences in their effective volume resulting from differences in their content of protein IIb. The most rapidly migrating band of this series contains virions with a few molecules of IIb protein, whereas the more slowly migrating bands contain virions with a larger number of IIb molecules. The adjacent bands in the series contain virions which differ by approximately three IIb molecules. Wild-type Qbeta virus is similar to the mutant in that the more slowly migrating virions contain more protein IIb than the more rapidly migrating virions. Their failure to resolve into distinct bands upon electrophoresis is believed due to a less restricted packing of protein IIb into their virions. Both wild-type Qbeta and mutant 27-2 also have 1 to 5% of the virions in the form of dimers which migrate with approximately one-half the mobility of the respective monomer forms. When the average amount of IIb per virion is increased by growth of the virus in a UGA suppressor strain, the electrophoretic pattern is altered. In the case of wild-type Qbeta, the single band is wider, whereas with Qbeta mutant 27-2 there occurs an increased number of partially resolved narrow bands. We suggest that the structural feature responsible for the difference in electrophoretic pattern between mutant 27-2 and wild-type Qbeta is the mode of IIb packing in the virions. In the mutant, the IIb proteins are found in the virions only in multiples of three, whereas wild-type virions may differ by only a single IIb protein.

Component four of brome mosaic virus RNA is a structural component of the virus but is not required for infection. Sequence analysis of oligonucleotides resulting from pancreatic ribonuclease digestion of the brome mosaic virus RNA's indicates that the nucleotide sequence of component four occurs in brome mosaic virus RNA component three but not in components one and two.

The RNA bacteriophage R17 when exposed to pH 10.5 is disrupted to yield a protein and products of alkaline hydrolysis of the viral RNA. The protein has an intrinsic sedimentation coefficient of 41.1 s and an intrinsic viscosity of 0.055 dl./g at 20 °C. It has a typical protein absorbancy spectrum and contains less than 1% RNA. These data, together with electron micrographs, indicate that the protein consists of a hollow protein shell similar in structure to the protein portion of R17.

Bromegrass mosaic virus undergoes a reversible decrease in its sedimentation coefficient when the pH is raised above pH 6.7. At pH 6 the sedimentation coefficient is 87 S, at pH 7 it is 79 S. Intrinsic viscosities determined at pH 6 and 7 are 3.64 and 5.5 x 10(-2) dl/gm. Diffusion coefficients are 1.56 x 10(-7) cm(2)/sec. and 1.44 x 10(-7) cm(2)/sec., respectively. Radii of gyration, measured by x-ray scattering, are 106 and 128 A. However, appropriate combination of sedimentation, diffusion, and viscosity coefficients at pH 6 and 7 yield the same molecular weight. Also, the zero-angle value of x-ray-scattered intensity, which is a function of molecular weight, is the same at the two pH's. These results suggest that bromegrass mosaic virus particles undergo a pH-induced change in structure. This change causes, among other things, an increase in the susceptibility of the particles to degradation by pancreatic ribonuclease. The shape of the titration curve between pH 6.3 and 6.9 is anomalous.

Features of the primary structure and translation of the genomic RNAs of the cowpea and bean strains of southern bean mosaic virus have been investigated in order to assess the similarity of the two viruses. The sequence of 400 bases at their 3' termini have been determined. These include the 3' noncoding regions and extend well into the coat protein cistrons. The noncoding regions (136 bases for the cowpea strain RNA and 129 bases for the bean strain RNA) show no obvious sequence homology. However, extensive base as well as amino acid sequence homology exists in the coding region. RNAs from both strains have a small protein attached to their 5' terminus-the protein in the cowpea strain being the smaller of the two. In vitro studies show that there are similarities in the overall mode of translation of the genomes of the two viruses. Although corresponding proteins are synthesized they differ in size.

THE ability of RNA from several plant viruses to bind a specific amino acid in a manner like tRNA is now well established. All members of the tymovirus group so far examined contain RNA which can bind valine1–3; all members of the bromovirus group bind tyrosine (ref. 4 and T.C.H., in preparation), and tobacco mosaic virus (TMV) RNA binds histidine5. The ubiquity of the reaction, and the similarity of the esterification to that of aminoacylation of tRNA indicates that a significant biological function for the property exists. A tRNA-like function such as that of a virus-specific tRNA, or a missense tRNA seems plausible. Although there is evidence that turnip yellow mosaic virus (TYMV) RNA, or a fragment containing the 3′ terminus of TYMV RNA, may donate valine to nascent polypeptides6, it has been established that tyrosine, bound to brome mosaic virus (BMV) RNA, is not donated to peptidyl material in a cell-free system from wheat embryo when directed by viral, plant, or synthetic messenger RNA (ref. 7).

Publisher Summary This chapter describes brome mosaic virus (BMV) as a small, multicomponent plant virus. It consists of three virions, designated H, L and M, which contain RNAs 1, 2, and 3-plus-4. RNAs 1, 2, and 4 are monocistronic messengers for proteins of molecular weight approximately 110,000, 105,000 and 20,000; the latter is BMV coat protein. RNA 3 is a dicistronic messenger; it encodes a protein of MW 35,000 and also coat protein. It reviews that all four RNAs are excellent messengers in cell-free extracts derived from wheat embryo. However, only the cistron for the 35,000 MW proteins is translated efficiently by RNA 3. All four RNAs have m 7 GpppG at the 5' terminus, and all four have nearly identical sequences extending for 160 nucleosides from their 3' terminus. The latter region is somewhat resistant to nucleases and can be obtained intact by partial digestion of the RNAs with ribonuclease T1. These 160-nucleoside fragments, as well as the RNAs from which they were obtained, are chargeable with tyrosine in reactions catalyzed by wheat-germ tRNA synthetases.

The four RNAs of the multicomponent Brome Mosaic Virus have been translated in wheat embryo cell-free extracts in order to characterize their cistrons. RNAs 1 and 2 are monocistronic; they each direct the synthesis of a single large polypeptide. RNA 3 is dicistronic but only one of its cistrons is translated. RNA 4 is a monocistronic messenger for the coat protein. The 5′ end of RNA 4 begins with m 7 G and a short sequence of A's and U's; these are followed by the initiation codon and codons for the amino acids of the coat protein. RNAs 1, 2 and 3 also have m 7 G at their 5′ terminus. The 3′ region of all four RNAs is remarkably similar in sequence.

In cell-free extracts derived from rabbit reticulocytes, encephalomyocarditis RNA can be translated completely, and the products can be processed extensively to give encephalomyocarditis virion proteins and several nonvirion proteins, including a genome-coded protein required for processing. The latter is probably a protease. Translation is very efficient. Under typical conditions, each EMC RNA is translated approximately eight times during a 3-h period. Kinetic analyses (time-course experiments, pulse-chase experiments, and pulse-stop experiments) have been used to determine the time of appearance of major products, and these times have been correlated with map positions. The gene for the putative protease is located near the middle of the genome downstream from the virion protein genes. Ribosomes can travel the length of encephalomycarditis RNA within 30 min, but there is a delay in their progress along the RNA at some point soon after they traverse the region coding for virion protein precursors. This delay results in the accumulation of precursors for a period of about 10 min before the putative protease is made and virion proteins (epsilon, alpha, and gamma) are released by proteolysis.

Each of the two largest brome mosaic virus RNAs, RNA1 and RNA2, directs the synthesis of a large protein in cell-free extracts derived from wheat embryo. The size of each protein represents the translation of almost the entire length of the corresponding RNA. It was shown previously that brome mosaic virus RNA4 directs the synthesis of the coat protein and that brome mosaic virus RNA3, although it also contains the coat protein cistron, is translated mostly into a single product unrelated to the coat protein (Shih & Kaesberg, 1973). Thus, the brome mosaic virus genome encodes a total of four proteins.

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration.

A complementary DNA copy of the 3a cistron of dicistronic RNA3 from brome mosaic virus has been cloned and sequenced. Based on this and other data, the complete nucleotide sequence of brome mosaic virus RNA3 is presented. Following a 91 base 5′ non-coding region, the 909 base 3a cistron encodes a protein of predicted molecular weight 32,480. Between the 3a and coat protein cistrons is an approximately 250 base intercistronic non-coding region. Overlapping the intercistronic region by 9 bases, the last 876 bases of RNA3 encode the subgenomic coat protein messenger, RNA4, completely and without interruption. A third long open reading frame of possible evolutionary significance overlaps the 3a and coat protein cistrons. An internal poly(A) of heterogeneous length (16 to 22 bases) occurs in the intercistronic region just 20 bases 5′ to the start of RNA4 sequences. Averaging the poly(A) heterogeneity, the length of brome mosaic virus RNA3 is 2114 bases.

The structure and size of potato spindle tuber viroid (PSTV) were investigated by electron microscopy. PSTV appears to be composed of two single-stranded RNA components of different sizes, one linear and one circular. The linear component is about four times more common than the circular. Both components exist in collapsed, highly base-paired structures under nondenaturing conditions. Both components are sensitive to digestion by pancreatic ribonuclease but are resistant to snake venom phosphodiesterase. From length measurements of formylated and non-formylated PSTV RNA, the estimated molecular weight for the linear molecule is 110,000, and for the circular, it is 137,000.