Outer capsid protein heterogeneity of rice dwarf phytoreovirus.

The 46K outer capsid protein encoded by RNA segment S8 and the 42K polypeptide, previously thought to be the segment S9-encoded structural protein, were isolated from a rice dwarf phytoreovirus purified preparation, and then analysed by peptide mapping and electroblot-ELISA. Staphylococcus aureus V8 protease peptide mapping patterns of the 42K and 46K proteins were similar. Two monoclonal antibodies (MAbs), obtained after immunization with virus particles dissociated by 0.1% SDS, were each specific for both the 42K and 46K proteins. Furthermore, the MAbs bound common peptide fragments which were generated by digestion of the 42K and 46K proteins with V8 protease or proteinase K. These results strongly suggest that the 42K protein is not a gene product of S9 but a product overlapping with the 46K outer capsid protein. Whether the two proteins are functionally distinct remains to be determined.

Crystallization and preliminary X-ray study of a double-shelled spherical virus, rice dwarf virus.

Rice dwarf virus (RDV) is a double-shelled spherical plant virus consisting of 46,000 Mr capsid and 114,000 Mr core proteins and minor structural proteins, and containing 12 genome segments of double-stranded RNA. The virus has been crystallized in the cubic space group I23 with a = 789 A. There are two particles per unit cell, each positioned on a point of 23 symmetry. Packing considerations showed that the diameter of the virus particle is 693 A. The crystals diffract to at least 6.5 A resolution.

Coat protein of potyviruses. 7. Amino acid sequence of peanut stripe virus.

The amino acid sequence of the 287-residue coat protein of peanut stripe virus (PStV) was determined from the sequences of overlapping peptide fragments. Results indicated that the amino terminus was blocked by an acetyl group, as has previously been found for the coat protein of Johnsongrass mosaic potyvirus. Comparison of the PStV sequence with coat proteins of 20 distinct potyviruses gave sequence identities of 47-57%, except for zucchini yellow mosaic virus (ZYMV), passionfruit woodiness virus (PWV), and the related strains watermelon mosaic virus 2 (WMV 2) and soybean mosaic virus-N, which showed sequence identities of 70-76%. Several amino acid residues which were common to the core sequences of these coat proteins were at positions previously found to be invariant among potyvirus coat proteins. The degree of these similarities suggests that although PStV, WMV 2, ZYMV, and PWV are distinct potyviruses, they share a common ancestor in their evolutionary development.

Purification of plant viruses and virus coat proteins by high performance liquid chromatography.

High performance liquid chromatography (HPLC) gel filtration has been successfully applied in the purification of elongated and isometric plant viruses. Two different approaches have been tested. In one approach, semi-purified virus particles were dissociated with lithium chloride and the released coat proteins purified by HPLC gel filtration. The purified coat protein was highly immunogenic and gave rise to very specific antisera reacting with intact virus particles as well as with SDS-denatured coat protein monomers. This method is generally applicable only for elongated viruses since many isometric viruses are not dissociated by the lithium chloride treatment. The second approach consisted in gel filtration of native, undissociated virus particles and could be used both with elongated and isometric viruses. Both methods were fast and simple to perform and removed all or most of the contaminating plant proteins as judged by sodium dodecylsulphate gel electrophoresis followed by silver staining or by immunoblotting with antiserum against healthy plant extracts. With both methods the recovery of virus coat protein was about 30% on average.

Similarities between putative transport proteins of plant viruses.

The nucleic acids of many plant viruses encode proteins with one or more of the following properties: an Mr of approximately 30,000, localization in the cell wall of the infected plant and a demonstrated role in cell-to-cell transport of infection. A progressive alignment strategy, aligning first those sequences known to be similar, and then aligning the resulting groups of sequences, was used to examine further the relatedness of the amino acid sequences of putative transport proteins of caulimoviruses, of proteins similar to the putative transport protein of alfalfa mosaic virus (A1MV) and of those similar to the tobacco mosaic virus (TMV) 30K protein. The strategy first identified regions in which multiple dipeptides of one group were similar to those of another group. The regions of similarity were brought into alignment by the conservative introduction of gaps. The positions of the introduction of gaps were adjusted to optimize similarity. Statistical significances of the resulting alignments, determined both by comparison with shuffled amino acid sequences and with the sequence alignment off-set by 1 to 15 residues in each direction, suggest that the amino acid sequences of the three groups of viruses are distantly related. Nevertheless, significant relationships between members of the caulimoviral group of sequences and members of each of the A1MV-like and TMV-like groups were found. These relationships and the analysis of the number of insertions/deletions between present sequences and a hypothetical common ancestor suggest that the sequences of the caulimoviral proteins are less diverged from the ancestor than either the A1MV-like or TMV-like proteins. The alignment identified common regions of predicted secondary structure and regions of similar hydropathy, regions possibly crucial for proper functioning of the proteins.

Sequential removal of Ca2+ from satellite tobacco necrosis virus. Crystal structure of two EDTA-treated forms.

Two forms of EDTA-treated satellite tobacco necrosis virus (STNV) have been studied with X-ray crystallography methods. The crystals of both forms were isomorphous with native STNV crystals, and (FEDTA-Fnat) maps as well as (2FEDTA-Fnat) maps were calculated with phases from the native structure. The maps were based on partial data sets to 2.8 A resolution, and averaged using the 60-fold non-crystallographic symmetry. In the first crystal form, calcium ions were absent from one of the three sites in the icosahedral protein shell. The crystals were produced at pH 5.0 from a virus solution treated with EDTA at pH 6.5. The virions were not expanded, and no essential changes were seen in the protein shell. In the second crystal form, all calcium ions in the protein shell were absent. The virus material in these crystals had been subjected to treatment with EDTA at pH 8.0 before crystallization at pH 6.5. The high pH treatment caused degradation of the viral RNA. No expansion of the virion had occurred and all protein--protein contacts were retained. These results are compared with the previously presented low-resolution structure of slightly expanded STNV with intact RNA, where calcium ions from two sites were absent. The relevance of Ca2(+)-depleted virions for infection in vivo is discussed as well as the possibility that the Ca2(+)-binding sites may be parts of ion channels in the viral capsid. One possible RNA-binding site was found in the maps of both crystal types, and the same site could also be localized in the high-resolution map of native STNV.

The cauliflower mosaic virus gene III product is a non-sequence-specific DNA binding protein.

The interaction of the gene III product, P15, of cauliflower mosaic virus with different double-stranded DNA fragments of the viral genome was investigated. The results suggest that gene III product which showed DNA binding activity is a structural protein of the viral particle.

Unusual electrophoretic properties of the coat protein of raspberry ringspot nepovirus.

Coat protein of raspberry ringspot nepovirus (RRV) migrated during electrophoresis in a discontinuous buffer system as two components which were capable of interchanging during this procedure. However, only one component was formed during electrophoresis in a continuous buffer system. Treatment of the protein before electrophoresis with iodoacetamide, which alkylates sulfhydryl groups, prevented the formation of the faster migrating electrophoretic component. Thus the faster migrating component is probably an artifact caused by S-S bond formation. Although the coat proteins of two other nepoviruses did not behave in this way, the results obtained with RRV emphasize the need for caution in interpreting electrophoretic heterogeneity in virus proteins. The estimated molecular weight of RRV coat protein was 57,000.

Identification and subcellular localization of a putative cell-to-cell transport protein from red clover mottle virus.

To investigate the mode of gene expression of red clover mottle virus (RCMV) middle component (M) RNA, we have synthesized an oligopeptide corresponding to the predicted carboxy-terminus of the RCMV counterparts of the cowpea mosaic virus (CPMV) 48K and 58K proteins. Using an antiserum raised against this synthetic oligopeptide, we have detected a 43-kDa protein in the 30,000 g pellet from extracts of RCMV-infected cowpea protoplasts. Immunogold cytochemistry further localized this protein to the plasmodesmata of RCMV-infected pea tissue. This subcellular location, taken together with other evidence, suggests that this 43-kDa protein has a role in the cell-to-cell spread of RCMV.

Partial cleavage of sweet potato feathery mottle virus coat protein subunit by an enzyme in extracts of infected symptomless leaves.

The coat protein of particles of sweet potato feathery mottle potyvirus (SPFMV) extracted from Ipomoea spp. migrated in SDS-PAGE mainly as bands of M(r) 38,000 (38K), 36K, 32K, 30K. Trypsin treatment of the particles resulted in the appearance of only one 30K polypeptide. The inclusion of protease inhibitors in the extraction procedure did not alter the heterogeneity of SPFMV coat protein. A partially purified fraction of extracts from recovering, symptomless, but not from healthy leaves of I. nil had a proteolytic activity similar to that of trypsin. Amino acid sequencing showed that the trypsin-cleaved 30K polypeptide had some sequence homology with other potyvirus coat proteins. The site at which the Ipomea extract cleaved the protein was five amino acids nearer the N terminus than trypsin cleavage site.

Production of antiserum to a non-structural potyviral protein and its use to detect narcissus yellow stripe and other potyviruses.

A protein, of apparent molecular weight 72,000, was purified from experimentally infected narcissus plants with yellow stripe symptoms utilising SDS-polyacrylamide gel electrophoresis. This protein was excised from the gels and used to prepare antiserum, which reacted specifically with cytoplasmic cylindrical inclusions in ultra-thin sections of virus-infected cells and, in immunoblots, with the 72 kDa protein in preparations containing cytoplasmic inclusions. The antiserum reacted in ELISA with leaf extracts from yellow stripe diseased plants of four narcissus cultivars but not with extracts from comparable symptomless plants. In tests with extracts of plants infected with seven definitive potyviruses, reactions were obtained with bean yellow mosaic and iris mild mosaic viruses. Virus-specific reactions in dot-blot ELISA were dependent on the presence of Tween 20 in the extraction buffer. In contrast, an antiserum to the putative cytoplasmic inclusion protein of alstroemeria mosaic virus reacted only with SDS-treated leaf extracts of infected plants. In limited tests, the method of purifying cytoplasmic inclusion protein was successfully applied to four definitive potyviruses, suggesting that it may be generally applicable to potyviruses and of use for preparing antisera when purification of virus particles is difficult.

Protein-RNA interactions in an icosahedral virus at 3.0 A resolution.

Nearly 20 percent of the packaged RNA in bean-pod mottle virus (BPMV) binds to the capsid interior in a symmetric fashion and is clearly visible in the electron density map. The RNA displaying icosahedral symmetry is single-stranded with well-defined polarity and stereochemical properties. Interactions with protein are dominated by nonbonding forces with few specific contacts. The tertiary and quaternary structures of the BPMV capsid proteins are similar to those observed in animal picornaviruses, supporting the close relation between plant comoviruses and animal picornaviruses established by previous biological studies.

Variability and evolution of the plant RNA virus pepper mild mottle virus.

The RNA genomes of 26 isolates of pepper mild mottle virus were compared by their RNase T1 fingerprints. Twenty-three isolates came from epidemic outbreaks in greenhouse-grown peppers in Almería (southeastern Spain) from 1983 to 1987; three other isolates, from 1980, came from Sicily (Italy) and Zaragoza (central Spain). The 26 fingerprints can be classified into 10 different types; nucleotide substitution rates show them to be very similar. Cluster and cladistic analyses group types corresponding to the Almería isolates separate from those of 1980. Intraannual and interannual nucleotide differences were estimated. An evolutionary model for pepper mild mottle virus built on these data indicates a highly stable population, maintaining its diversity through time, with a main prevailing haplotype from which closely related variants arise that do not replace it. This high stability could be due to strong functional constraints on variation, as suggested by the high proportion of invariant versus polymorphic sites in fingerprints.

Primary structure of belladonna mottle virus coat protein.

The coat protein of belladonna mottle virus (a tymovirus) was cleaved by trypsin and chymotrypsin, and the peptides were separated by high performance liquid chromatography using a combination of gel permeation, reverse phase, and ion pair chromatography. The peptides were sequenced manually using the 4-N, N-dimethylaminoazobenzene-4'-isothiocyanate/phenyl isothiocyanate double-coupling method. The chymotryptic peptides were aligned by overlapping sequences of tryptic peptides and by homology with another tymovirus, eggplant mosaic virus. The belladonna mottle virus is more closely related to eggplant mosaic virus than to turnip yellow mosaic virus, the type member of this group, as evident from the sequence homologies of 57 and 32%, respectively. The accumulation of basic residues at the amino terminus implicated in RNA-protein interactions in many spherical plant viruses was absent in all the three sequences. Interestingly, the amino-terminal region is the least conserved among the tymoviruses. The longest stretch of conserved sequence between belladonna mottle virus and eggplant mosaic virus was residues 34-44, whereas it was residues 96-102 in the case of belladonna mottle virus and turnip yellow mosaic virus. A tetrapeptide in the region (residues 154-157) was found to be common for all the three sequences. It is possible that these conserved regions (residues 34-44, 96-102, 154-157) are involved in either intersubunit or RNA-protein interactions.

Comparison of proteins and nucleic acids of Echinochloa ragged stunt and rice ragged stunt viruses.

Echinochloa ragged stunt virus (ERSV) and rice ragged stunt virus (RRSV; Reoviridae) were purified from their respective host plants and disrupted in SDS. Evidence for the double-strandedness of ERSV RNA was obtained. Electrophoresis in 10% polyacrylamide gels resolved the RNAs of each virus into 10 segments, ranging for ERSV from 0.61 x 10(6) to 2.8 x 10(6) daltons, with a sum representing the total genome of 17.89 x 10(6) daltons, and for RRSV from 0.6 x 10(6) to 2.98 x 10(6) daltons, with a sum of 18.15 x 10(6) daltons. Although the overall RNA pattern of the two viruses was similar, there were distinctive features. Using 7.5-15% polyacrylamide gels, electrophoresis of SDS-dissociated RRSV particles yielded five major proteins of 129 x 10(3), 123 x 10(3), 63 x 10(3) and 35 x 10(3) daltons, and two minor proteins of 113 x 10(3) and 88 x 10(3) daltons, respectively, while comparable samples of ERSV yielded four major proteins of 127 x 10(3), 123 x 10(3), 63 x 10(3) and 34 x 10(3) daltons, and three minor proteins of 103 x 10(3), 50 x 10(3) and 49 x 10(3) daltons, respectively. We conclude from this and other evidence that ERSV and RRSV are closely related viruses and should be classified in the same subgroup, despite reported differences in the morphology of their outer capsids.

Virus-ribosome complexes from cell-free translation systems supplemented with cowpea chlorotic mottle virus particles.

When particles of cowpea chlorotic mottle virus (CCMV) were added to cell-free extracts from wheat germ, the encapsidated viral genome was translated into polypeptides similar to the translation products specified by unencapsidated viral RNA (as shown before by M.J. Brisco, R. Hull, and T.M.A. Wilson, 1986, Virology 148, 210-217). The rate of protein synthesis observed upon addition of virus particles was much slower than that of extracted RNA and the quantity of protein formed was only 10% of that of extracted RNA. Using sucrose and cesium-chloride gradient analysis, virus-ribosome complexes, containing up to four ribosomes per virus particle, were isolated from translation mixtures supplemented with CCMV particles. These complexes, with densities intermediate of those of virus (1.36 g cm-3) and ribosomes (1.58 g cm-3), were analyzed and quantified in the electron microscope. Less than 5% of the particles was found in association with ribosomes. To verify whether these complexes were involved in the process of cotranslational disassembly, tobacco mosaic virus was analyzed with the same techniques and methods. The results found for TMV were similar to those found for CCMV except that virus-ribosome complexes with up to 20 ribosomes per virus particle were observed. The implications of the process of virion-directed translation for the structure of the particle as well as the role of this process in vivo are discussed.

Relationship of lychnis ringspot virus to barley stripe mosaic virus and poa semilatent virus.

Barley stripe mosaic virus (BSMV), poa semilatent virus (PSLV), and lychnis ringspot virus (LRSV) have previously been assigned to the hordeivirus group because of similarities in their particle morphology, physicochemical properties and serological analyses. However, the serological relationships of the three viruses have not been determined by direct comparison. The present study evaluated the relatedness of these viruses by Western and dot immunoblotting and by nucleic acid hybridizations. Serological analyses of the coat proteins separated by gel electrophoresis and of intact virus particles bound to nitrocellulose membranes revealed that BSMV and PSLV are distantly related, but that they are more closely related to each other than to LRSV. The genomic RNAs of the viruses failed to cross-hybridize in northern hybridization tests conducted at different temperatures. These comparisons showed that BSMV, PSLV and LRSV are distinct viruses with little nucleotide sequence relatedness. Thus our data provide additional support for their inclusion as separate members of the hordeivirus group.

Detection of turnip crinkle virus on agarose gel electropherograms at the nanogram load level.

The previous conditions for the physical characterization of turnip crinkle virus (TCV) by quantitative agarose gel electrophoresis [1, 2] were limiting the method to the microgram load level and were therefore insufficiently sensitive to satisfy the need in many areas of virology for detection of viruses containing single-stranded RNA at the nanogram level. The present report remedies that defect by presenting a technique compatible with the nanogram load level of such viruses. The technique is based on a reduction of gel thickness and on the use of silver staining.

Physical identification of a virus in a crude leaf extract by its Ferguson plot in agarose gel electrophoresis.

Crude extracts of turnip crinkle virus upon agarose gel electrophoresis yield (i) virus patterns unperturbed by contaminants; (ii) plots of mobility vs. gel concentration (Ferguson plots) parallel with those of the purified virus. The parallelism suggests similarity in size and shape but a lower net charge for the crude virus. This result is obtained when gel electrophoresis is carried out either in a continuous buffer or in a discontinuous (moving boundary electrophoresis) buffer system. The latter mode has the substantial benefit of electrophoretic (auto-)concentration of dilute virus sample prior to resolution. Thus, the Ferguson plot analysis in a discontinuous buffer system of turnip crinkle virus can be viewed as a model procedure for the physical identification of other viruses contained in dilute extracts, feasible even in the absence of a prior knowledge as to the nature of, or isolation of, the virus.