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The 3' proximal portion of the gene encoding the 201-kDa putative replicase protein from the Tobravirus pea early browning virus (PEBV) can potentially be expressed separately as a 54-kDa protein. Nicotiana benthamiana plants transformed with the open reading frame (ORF) encoding the 54-kDa protein, designated 54K ORF, were resistant to infection by purified PEBV at inoculum doses of up to 1 mg/ml, the highest concentration tested. However, resistance was abolished by the introduction into the 54K ORF of mutations that would cause premature termination of translation. This suggests that the resistance mechanism requires the involvement of an intact 54-kDa protein. The 54K ORF-transformed plants were also resistant to infection by broad bean yellow band virus and an uncharacterized isolate of British PEBV (PGRO R) but were not resistant to infection by two other tobraviruses, pepper ringspot virus and the I6 isolate of tobacco rattle virus. Additionally, two variants of PEBV which overcame 54K ORF-mediated resistance have been isolated, the analysis of which might provide important information about both the resistance mechanism itself and the process of normal virus replication.

Comparison of the 5'-terminal sequences of several tobraviruses suggests that the RNA-2 molecule of the tobacco rattle virus (TRV) anomalous isolate TCM arose from pea early browning virus (PEBV) RNA-2 by acquisition of 3' and 5' sequences from TRV RNA-1 and RNA-2 molecules, respectively. We have identified a region of homology in the RNA-2 molecules of PEBV, TRV and pepper ringspot virus which could have facilitated this recombination.

The complete nucleotide sequence of the middle component RNA (M RNA) of the comovirus bean pod mottle virus (BPMV) has been determined. The sequence consists of 3662 nucleotides and contains a single long open reading frame sufficient to code for a protein of 113,353 Da. The proteolytic processing sites within this protein have been identified by comparison with the known three-dimensional structure of the virion and cleavage at these sites would lead to a range of products consistent with those observed during processing of the M RNA-encoded polyproteins in vitro. We have performed computer-aided searches for reiterated sequences within BPMV M RNA which might explain why ordered RNA is visible in the electron density map of BPMV middle component particles (Chen, Z., Stauffacher, C. V., Li, Y., Schmidt, T., Bomu, W., Kamer, G., Shanks, M., Lomonossoff, G., and Johnson, J. E., 1989, Science 245, 154-159). These searches revealed both the presence of overrepresented pentameric sequences and a consensus sequence which was repeated 15 times within the RNA sequence.

Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.

We have determined the sequence of 80 nucleotides adjacent to 3' poly(A) of both (middle and bottom component) cowpea mosaic virus RNAs, using dideoxynucleotide termination of reverse transcription. Sequence conservation is indicated, there being about 80% homology between the first 65 bases of each RNA. Although both RNAs are polyadenylated, there is no AAUAAA sequence as associated with most known polyadenylated mRNAs of eukaryotes and viruses. However both RNAs are U and A-rich in this region.

Narcissus-mosaic-virus RNA is translated into a coat-protein-sized product in wheat-germ cell-free extracts. This protein was shown to be very similar to authentic coat protein by partial proteolysis in SDS/polyacrylamide-gel electrophoresis, and by serology. Fractionation of the RNA revealed a small RNA molecule of approx. 840 nucleotides, which alone coded for the coat protein. This subgenomic RNA was found to be encapsidated in a short virus particle.

The multipartite nature of the brome mosaic virus genome is described. Particular attention is given to the translation of the RNAs in cell-free extracts, from dry wheat seed embryos and commercial wheat germ. Such studies have demonstrated that the smallest RNA is a monocistronic message for coat protein. This cistron is also carried on the intermediate sized RNA, which also codes for a larger protein, of unknown function. Translation of the larger RNAs 1 and 2 is compared, incell-free extracts of wheat embryo and wheat germ. There is evidence that these RNAs may also be monocistronic messages for large polypeptides (molecular weight 100,000), indicating that there are few non-translatable regions on these RNAs. Some recent nucleotide sequence studies are described, including the ribosome binding site of the coat protein cistron, and the 3' termini of all four RNAs, which appear to be identical, at least over a region of 150 nucleotides.

Evidence is presented for the presence of a protein covalently bound to the 5' termini of both M and B RNA components of CPMV. The protein is found to be linked in both cases to the 5' phosphate of the dinucleotide pUpAp, derived by ribonuclease digestion of the RNA. The intact protein is not required for infectivity or for in vitro translation of the RNA in cell-free extracts.

We have determined the complete nucleotide sequence of RNA1 of the tobravirus pea early browning virus [PEBV] from an overlapping series of cDNA clones. The 7073 nucleotide sequence contains four open reading frames [ORFs]. The 5' proximal ORF encodes a 141K polypeptide, and readthrough of the opal [UGA] termination codon of this ORF would lead to the synthesis of a second, 201K polypeptide. Both of these polypeptides have extensive amino acid homology with the putative replicase proteins of tobacco rattle virus [TRV] and tobacco mosaic virus [TMV]. The third ORF encodes a 30K polypeptide which has homology with the TRV 29K and TMV 30K putative cell-to-cell spread proteins. The fourth, 3' proximal ORF encodes a 12K polypeptide which has extensive homology with the TRV 16K protein whose function is unknown. Examination of the amino acid sequences of the 12K and 16K gene products reveals in each the presence of two multiple-cysteine/histidine motifs, a finding which suggests that these proteins might have zinc and/or nucleic acid-binding properties.

Conditions are described which give an efficient synthesis of DNA copies of cowpea mosaic virus (CPMV) RNAs, using avian myeloblastosis reverse transcriptase and oligo (dT) primers. Maximum incorporation of dAMP into cDNA is attained with 0.4 to 0.8 mM of each deoxynucleoside triphosphate, 12 mM Mg++ and 60 mM K+ ion concentrations. High enzyme concentrations (up to 100 units/ml) were used. Under these conditions over 1000 pmoles of dAMP were incorporated per reaction. The cDNA:RNA molar ratio approached 0.3 when 1 pmole CPMV RNA was used as template. The products were heterogeneous but large. Bottom component RNA (about 6000 nucleotides long) was copied into cDNA molecules ranging from about 1000 to 4000 nucleotides, and middle component RNA (about 4000 nucleotides long) was copied into cDNA mostly between 500 and 2000 nucleotides long, on average about 1500, which can be cleaved by restriction endonuclease Hae III into two fragments of 880 and 540 nucleotides.

The RNA from bacteriophage Qbeta can be translated by cell-free extracts from wheat embryos. This translation, by 80S ribosomes, occurs at a low magnesium ion concentration. Three products are synthesized which coelectrophorese with Qbeta 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.