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The sequences of the 5′ terminal 1140 and 3′ terminal 1546 nt of tomato ringspot virus (TomRSV) RNA1 have been determined. These sequences share a high degree of nucleotide sequence similarity with the previously determined TomRSV RNA2 sequence. Eighty-eight percent of the 5' terminal 907 nt of TomRSV RNA1 and RNA2 contain identical nucleotide residues; the first 459 nt are identical at all positions, whereas the next 447 nt are identical at only 75.8% of the nucleotide positions. The region of similarity includes not only the 5' nontranslated leader but also sequence probably encoding polyproteins. The 3′ terminal 1533 nt of TomRSV RNA1 and RNA2 are identical and are noncoding. The sequences common to RNA1 and RNA2 account for almost 35% of the total genomic sequence. It is possible that the similar sequences at both ends of TomRSV RNA1 and RNA2 are a result of recombination between these two genomic RNA components.

The coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection by the ME strain of PVS as indicated by an absence of symptom development and a lack of accumulation of virus in both the inoculated and upper leaves. As in reported experiments with plants expressing potato virus X coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein-mediated protection for these two groups of viruses, which share similar genome organizations, may involve inhibition of some early event in infection other than or in addition to virus uncoating.

The sequence of tomato ringspot virus (TomRSV) RNA-2 has been determined. It is 7273 nucleotides in length excluding the 3′ poly(A) tail and contains a single long open reading frame (ORF) of 5646 nucleotides in the positive sense beginning at position 78 and terminating at position 5723. A second in-frame AUG at position 441 is in a more favourable context for initiation of translation and may act as a site for initiation of translation. The TomRSV RNA-2 3′ non-coding region is 1550 nucleotides in length. The coat protein is located in the C-terminal region of the large polypeptide and shows significant but limited amino acid sequence similarity to the putative coat proteins of the nepoviruses tomato black ring (TBRV), Hungarian grapevine chrome mosaic (GCMV) and grapevine fanleaf (GFLV). Comparisons of the coding and non-coding regions of TomRSV RNA-2 and the RNA components of TBRV, GCMV, GFLV and the comovirus cowpea mosaic virus revealed significant similarity for over 300 amino acids between the coding region immediately to the N-terminal side of the putative coat proteins of TomRSV and GFLV; very little similarity could be detected among the non-coding regions of TomRSV and any of these viruses.

Summary The sequence of 3553 nucleotides corresponding to the 3′-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames (ORFs) encoding proteins of M r 10734, M r 32515, M r 7222, M r 11802, M r 25092 and at least M r 41052. The sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from plasmids which were isolated by colony hybridization with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lystates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59% identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORFs displayed significant sequence homology with the similarly sized ORFs from a number of potexviruses. The partial 41K ORF product was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus.

Summary Four isolates of carnation ringspot virus showed a reversible aggregation which was dependent on the temperature and concentration of their preparations. At 7 mg/ml, preparations of two of these isolates aggregated at room temperature but at the same concentration the other two isolates required higher temperatures (40 °C) for aggregation. A fifth isolate formed aggregates of 12 virus particles and linked aggregates. Particles of the two isolates with less tendency to aggregate were almost completely dissociated by 0.4% sodium dodecyl sulphate (SDS) at pH 5 and by 0.1% SDS at pH 7. At pH 7, an RNA component of mol. wt. 0.5 × 106 was released by lower concentrations of SDS than was an RNA component of 1.5 × 106. At pH 5, the sedimentation rates of unaggregated particles of the remaining three aggregating strains were unaffected by up to 15% SDS. However, treatment with 0.0075 to 0.05% SDS at pH 7 produced protein, RNA and swollen virus particles. These swollen particles were not further affected by increasing the SDS concentration to 8%.

Summary The N-terminal cyanogen bromide peptide of the coat protein of the cowpea strain of southern bean mosaic virus (SBMV-C) was purified by high-pressure liquid chromatography and used as an immunogen in the production of monoclonal antibodies. One monoclonal antibody, designated 4D6, bound to both purified peptide and immobilized virus in a solid-phase ELISA. The reactivity of this monoclonal antibody with immobilized peptide could not be inhibited by native SBMV-C but was inhibited by low levels of EDTA-swollen virus in an antigen inhibition ELISA. The binding of 4D6 to the swollen conformation of the virus was not significantly diminished upon collapsing the swollen virus by pH adjustment or addition of calcium ions. Reactivity of the antibody was also observed with native virus particles which had been dialysed against weakly alkaline buffers. The binding of 4D6 was extremely sensitive to trypsin proteolysis of the virus coat protein, indicating that the antibody binding site was most likely located within the first 30 amino acid residues of the N-terminus. Monoclonal antibody 4D6 also cross-reacted weakly with red clover necrotic mosaic virus in both indirect ELISA and antigen inhibition ELISA.

Values of the equilibrium dissociation constant and the number of attachment sites for the binding of radiolabeled F AB fragments prepared from four monoclonal antibodies to southern bean mosaic virus have been determined. Monoclonal antibodies B6 and B10 produced against the bean type strain of southern bean mosaic virus (SBMV-B) and 1C4 and 1D6 produced against the cowpea type strain (SBMV-C) were more reactive with the untreated virus than with EDTA-swollen particles. B6 and B10 reacted with 180 and 90 sites, respectively, on the SBMV-B particle and were not reactive with SBMV-C. Antibody 1D6 reacted with 180 sites on both SBMV-B and SBMV-C, whereas 1C4 bound to 180 sites on SBMV-C and 60 sites on SBMV-B. The equilibrium dissociation constants for B10, 1C4, and 1D6 were similar but antibody B6 bound with an 8- to 10-fold lower affinity. Solid phase competitive antibody inhibition analysis showed that 1C4 and 1D6 were mutually inhibitory and that these antibodies also inhibited the binding of B6 and B10. Antibody B6 partially inhibited the binding of 1D6 but did not affect 1C4 or B10 binding. No inhibition of B6, 1C4, or 1D6 binding was observed when B10 was the competing antibody. Possible sites of reaction of these monoclonal antibodies on the SBMV particle are discussed.

The reactivity of six monoclonal antibodies with native southern bean mosaic virus particles, swollen virus, swollen virus contracted by divalent cations or by pH adjustment to 5.0, and virus coat protein at pH 7.5 and 5.0 were assessed by antigen inhibition enzyme-linked immunosorbent assays. Clones B4, B7, and B11 did not react with native virus but were inhibited by low levels of all nonnative virus antigens. B6 had a high reactivity only with the native virus. B5 and B10 reacted equally with native virus and swollen virus contracted by Ca2+ or by pH adjustment, but reacted only weakly with swollen virus or swollen virus contracted by Mg2+. B5 reacted with viral protein at pH 5.0 and weakly with coat protein at pH 7.5, whereas B10 did not react with protein at pH 7.5 and had only limited reactivity with viral protein at pH 5.0. The reactivity of B5 and B10 with swollen virus increased as the pH was gradually decreased between 7.3 and 6.5. Similarly, their reactivity with swollen-contracted virus decreased as the pH was increased within the same range. Reactivity versus pH plots of these two transition forms showed hysteresis with B10 but did not with B5.

Summary Cloned DNA representing the sequence coding for the non-structural protein 3A from cucumber mosaic virus (CMV) was inserted in an expression vector containing a truncated portion of the Protein A gene from Staphylococcus aureus. Expressed fusion protein was purified from Escherichia coli cell extracts by affinity chromatography on rabbit gamma globulin-conjugated Sepharose and used as an immunogen for the production of monoclonal antibodies (MAbs). Five MAbs that reacted with pXCM3A13 fusion protein in solid phase ELISA were able to immunoprecipitate specifically protein 3A from mixtures of in vitro translation products of CMV RNA. None of these antibodies reacted with the analogous protein in translation products of brome mosaic virus RNA, but all of them reacted with the 3A protein from tomato aspermy virus. Immunogold labelling of ultrathin sections of CMV-infected tobacco tissue with MAb 3H12 demonstrated that the 3A protein accumulated within the nucleoli of these cells. This location differs from that of the analogous 3A protein of alfalfa mosaic virus which is associated with the middle lamella of the cell walls of infected tobacco cells.

Summary Double-stranded cDNA copies of RNA-1 and RNA-2 of tomato ringspot virus were cloned into pUC9. Comparison of restriction enzyme maps of eight clones indicated that 1.9 kb at the 3′ termini of RNA-1 and RNA-2 were similar. Five of these clones were used in Northern hybridization analyses and found to contain sequences either unique to RNA-1 or RNA-2, or common to RNA-1 and RNA-2. Southern blot analyses using clones derived from RNA-1 and RNA-2 confirmed that there is a 1.9 kb nucleotide sequence homology at the 3′ termini. A sequence homology of this magnitude has not been reported previously for other plant viruses with multipartite ssRNA genomes.

Southern bean mosaic virus (SBMV) was swollen by treatment with EDTA at pH 7.5 and dissociated into RNA and protein in 1 M NaCl. Aliquots of this preparation were diluted with appropriate buffers to obtain samples in varying concentrations of NaCl, and components of these samples were sedimented through sucrose solutions and dissolved in 0.01 M Tris-HCI buffer, pH 7.5. The protein content and sedimentation properties of components in these preparations were determined. When the NaCl molarity in the treatment exceeded 0.6 M the preparations contained RNA with approximately six protein subunits per SBMV RNA molecule. The protein content of the preparations increased from 30 protein subunits per RNA molecule to 145 protein subunits per RNA molecule as the NaCl molarity used in the treatment was decreased from 0.5 to 0.1 M. The positions of sedimentation of components in these preparations in density gradient centrifugation were intermediate between those of RNA and EDTA-swollen virus. The sedimentation rate of these assembled components increased as the NaCl molarity used in the treatment was decreased. Similar components were assembled when preparations of RNA and protein dissociated from SBMV by dialysis in neutral buffers containing EDTA and 1 M NaCl were diluted to lower NaCl molarities. When SBMV was swollen by treatment with EDTA and dissociated in various concentrations of NaCl, the components formed were similar to those obtained by assembly in the same NaCl molarities. Preparations in the pH 7.5 buffer contained single components which sedimented at 56 S, 55 S, 54 S, 51 S, 46 S, 38 S, 33 S, and 24 S. With the exception of the 24 S component, components formed by disassembly in the same NaCl molarities and dissolved in pH 5.0 buffer sedimented faster.

Summary The amino acid composition, behaviour in SDS-polyacrylamide gel electrophoresis and electrophoretic patterns of cyanogen bromide peptides were studied for the protein subunits of different preparations of potato virus X (PVX). The results indicate that the protein subunits of PVX can be partially degraded in the intact virus at the N-terminus by reducing agent-dependent proteases in crude plant sap and by trypsin, and at the C-terminus by reducing agent-independent proteases occurring in some virus preparations.

The proteins of turnip rosette virus (TRosV) a sobemovirus, and the Prunus strain of tomato bushy stunt virus (TBSV-P) were cleaved with cyanogen bromide, and highly basic peptides were isolated by ion-exchange chromatography. The amino acid composition and sodium dodecyl sulphate – polyacrylamide gel electrophoresis indicated the basic peptide from TRosV had 40 amino acid residues, including 11 basic residues. This peptide had a composition similar to basic peptides isolated from the sobemoviruses, sowbane mosaic virus, and two strains of southern bean mosaic virus. The amino acid composition and size of the basic peptide from TBSV-P were similar to those found for the amino terminal sequence of the type strain of TBSV.

Cherry yellow virus, isolates Y.2 and Y.4, and necrotic ring spot virus, isolates N.4 and N.5, purified either from cucumber leaves or from sour cherry petals, were characterized by antigenically related particles that sedimented at approximately 72 S. Some preparations of each virus derived from either host also contained either a 35 S or a 22 S component usually having a low nucleic acid content. Such preparations were shown by the agar gel double-diffusion technique to contain two antigens, V and Q, that were only distantly, if at all, related. The 72 S component is associated with the V antigen, whereas the 22 S and 35 S components are tentatively considered to be two forms of the Q antigen. The Q antigen could also be detected in clarified expressed sap of infected cucumber tissue, but not in that of healthy cucumber nor in extracts prepared from healthy sources by methods used to purify the viruses. The Q antigen is thus associated with infection, but its origin has not yet been satisfactorily determined.

Concentrated extracts of petals from trees infected with stone-fruit virus diseases, and of cucumber plants inoculated with viruses isolated from these trees, were tested serologically by the gel-diffusion technique with antisera prepared by injection of petal and cucumber plant preparations into rabbits. An antigen, antigen Q, was detected in two yellows, latent and recurrent necrotic ring spot, two green ring mottle isolates from sour cherry, a prune dwarf isolate, two tatter leaf of sweet cherry isolates, and isolates from latent infections of plum and peach. Another antigen, the V antigen, was detected in all the isolates with the exception of a latent necrotic ring spot isolate. A third antigen, antigen P, was detected only in the two yellows, recurrent necrotic ring spot, two green ring mottle isolates from sour cherry, a prune dwarf isolate, and two tatter leaf isolates from sweet cherry. Some differences between the Q antigens in certain isolates were demonstrated, but the V antigens differed little from isolate to isolate. The recurrent necrotic ring spot isolate was shown to contain at least two distinct viruses and the P antigen was associated with one of these viruses.

Virus entities in cucumber associated with cherry yellows, green ring mottle, and necrotic ring spot of sour cherry; tatter leaf of sweet cherry; and prune dwarf were found to be serologically related but not identical. The virus antigens were compared in gel-diffusion precipitin tests with antisera prepared from both cucumber leaves and infected cherry petals.

The separation of a carnation virus from smaller cowpea-host antigens was achieved by column chromatography on a strongly basic ion-exchange resin. The separation was demonstrated by infectivity and serological tests, analytical ultracentrifugation, and ultraviolet spectrophotometry. The method is proposed for the removal of similar host components from preparations of other spherical plant viruses.

Summary Complementary DNA copies of the genomic RNA of melon necrotic spot virus (MNSV) have been cloned and the region deduced to encode the coat protein has been sequenced. The putative coat protein coding region, located near the 3′ end of the genome, consists of 1170 nucleotides and has the potential to encode a 390 amino acid protein of M r 41840. Our data show that although MNSV is a carmovirus, its coat protein more closely resembles those of the tombusviruses than those of the carmoviruses sequenced to date, in both the extent of sequence similarity and in the length of the random/arm and protruding domains of the coat protein. Furthermore, dot matrix comparisons revealed sequence similarity between the coat protein protruding domains of MNSV and the cucumber necrosis tombusvirus. This similarity may be involved in one or more of the biological properties these two viruses share, such as the ability to infect cucumbers naturally and to be transmitted by the soil-inhabiting fungus Olpidium radicale.

The protein of sowbane mosaic virus was cleaved with cyanogen bromide (CNBr) and a highly basic peptide, sCB-1, was isolated by Sephadex and ion-exchange chromatography. The amino acid composition and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that sCB-1 had 49 amino acid residues. Eighteen of these residues are basic, fifteen are lysine, and three are arginine. sCB-1 moved faster in polyacrylamide gel electrophoresis at pH 2.2 than a similar sized CNBr peptide from the bean strain of southern bean mosaic virus with twelve basic residues. The highly basic nature of sCB-1 suggests that this portion of the SoMV protein sequence is involved in protein-RNA binding in the virus particles.

Summary Antibodies induced by the injection of alfalfa mosaic virus (AMV) or the protein of turnip crinkle virus (TCV) reacted with the viruses and their respective protein subunits in gel-diffusion tests. The ratios of virus-reactive antibodies to protein-reactive antibodies were different in two fractions obtained by chromatography of the sera on DEAE-Sephadex. The first DEAE-Sephadex fraction contained a greater proportion of virus-reactive antibodies than did the second fraction or the unfractionated antiserum. Electrophoretic mobility measurements at pH 8.6 showed that AMV and TCV had a greater negative charge per unit surface area than did their respective proteins.

The molecular weight of Tipula iridescent virus, based on sedimentation and diffusion coefficients, was 5.51 × 10 8 , with hydration of 0.57 g of water per g of virus. Deoxyribonucleic acid content, based on total inorganic phosphorus liberated, was 19 ± 0.2%. At 260 mμ, the virus gave an uncorrected absorbance of 18.2 cm 2 /mg of virus and a light-scattering corrected absorbance of 9.8 cm 2 /mg of virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus. The possibility is discussed that the four iridescent insect viruses reported to date bear a strain relationship.