Refined structure of desmodium yellow mottle tymovirus at 2.7 A resolution

Desmodium yellow mottle virus is a 28 nm diameter, T=3 icosahedral plant virus of the tymovirus group. Its structure has been solved to a resolution of 2.7 A using X-ray diffraction analysis based on molecular replacement and phase extension methods. The final R value was 0.151 (R(free)=0.159) for 134,454 independent reflections. The folding of the polypeptide backbone is nearly identical with that of turnip yellow mosaic virus, as is the arrangement of subunits in the virus capsid. However, a major difference in the disposition of the amino-terminal ends of the subunits was observed. In turnip yellow mosaic virus, those from the B and C subunits comprising the hexameric capsomeres formed an annulus about the interior of the capsomere, while the corresponding N termini of the pentameric capsomere A subunits were not visible at all in electron density maps. In Desmodium yellow mottle tymovirus, amino termini from the A and B subunits combine to form the annuli, thereby resulting in a much strengthened association between the two types of capsomeres and an, apparently, more stable capsid. The first 13 residues of the C subunit were invisible in electron density maps. Two ordered fragments of single-stranded RNA, seven and two nucleotides in length, were observed. The ordered water structure of the virus particle was delineated and required 95 solvent molecules per protein subunit.     

Structure and Infectivity of Picornaviral RNA Encapsidated by Cowpea Chlorotic Mottle Virus Protein

Poliovirus and Mengo virus RNA were shown to associate efficiently with cowpea chlorotic mottle virus protein to form pseudovirions. The sedimentation coefficient for the pseudovirions was similar to that of poliovirus, and electron microscope observations showed the Mengo pseudovirions to be similar in size to Mengo virus. Such pseudovirions were infectious and were more resistant to ribonuclease than viral RNA; however, under our assay conditions, their infectivity was about equal to that of viral RNA.     

Near identity of 3′ RNA secondary structure in bromoviruses and cucumber mosaic virus

The 3′ terminal sequences of RNAs 1, 2, 3 and 4 from each of the three bromoviruses (brome mosaic, cowpea chlorotic mottle and broad bean mottle viruses) and also from cucumber mosaic virus display interviral sequence similarity in addition to strong intraviral homology. Interviral similarity is much more evident when RNA secondary, rather than primary, structures are compared. The last 190 bases of the various RNAs can fold into strikingly similar, extensively base-paired secondary structures whose common features are supported by RNA structure mapping. The extreme 3′ end of each viral RNA can base-pair in two distinct configurations. Bromovirus RNA 3s each contain an unusually accessible internal oligo(A) sequence which, in brome mosaic virus at least, is located in the intercistronic noncoding region. Functional implications of these structural features are discussed.     

Structure-based rationale for the rescue of systemic movement of brome mosaic virus by spontaneous second-site mutations in the coat protein gene.

We describe spontaneous second-site reversions within the coat protein open reading frame that rescue the systemic-spread phenotype and increase virion stability of a mutant of brome mosaic virus. Based on the crystal structure of the related cowpea chlorotic mottle virus, we show that the modified residues are spatially clustered to affect the formation of hexamers and pentamers and therefore virion stability.     

Effect of cordycepin triphosphate on in vitro RNA synthesis by plant viral replicases

In vitro RNA synthesis by tobacco mosaic virus and cowpea chlorotic mottle virus replicase were inhibited by cordycepin triphosphate. Inhibition could be overcome with higher concentrations of ATP in assay mixtures but not with UTP. Products synthesized in vitro by tobacco mosaic virus RNA replicase in the presence of inhibitor revealed replicative form but not replicative intermediate RNAs. These results suggest that cordycepin triphosphate competes specifically with ATP and results in premature termination of viral RNA synthesis in vitro.     

Peanut Chlorotic Ringspot Virus (PCRV), a Newly Discovered Virus Infecting Peanut (Arachis hypogaea L.)

A virus causing wide chlorotic ringspot (PCRV) associated with chlorotic line pattern and motthng on an Erictoides hybrid growing in USDA-OSU greenhouses, Stillwater, Oklahoma, was discovered. The virus was isolated and characterized and found to differ in symptomology, host range and serological properties from all the previously described viruses infecting peanut, particularly those reported in the United States to be the most important ones, peanut mottle virus, peanut stripe virus, and tomato spotted wilt virus. The virus was transmitted by both mechanical inoctilation and grafting to fourteen peanut cultivars causing identical symptoms to those originally observed on the Erictoides hybrid. In addition to peanut, the virus systemically infected Pisum sativum L. ‘Little marvel’ causing mainly mosaic and Lupinus albus L. ‘Tiftwhite’ producing severe malformation and remarkable reduction in leaflet area. The virus did not infect many other plant species of which cowpea ‘California blackeye’ (Vigna unguiculata L.) and at least five cultivars of soybean (Glydne max L.) are known to be susceptible hosts to peanut mottle virus. Phaseolus vulgaris L. ‘Topcrop’ and Chenopodium amaranticohr Coste & Reyn were found to be two useful local lesion assay and diagnostic hosts for PCRV. The virus elicited necrotic local lesions on the first and chlorotic ringspots on the second. PCRV had a dilution end point between 10^?5 and 10^?6, thermal inactivation point between 55°C and 60°C, and longevity in vitro up to 6 days but not 7 days. Virus particles viewed hy electron microscopy and the negative stain uranyl aceute were flexuous filamentous particles ranging in length from 750–850 nm. In both indiren PAS-ELISA and Ouchterlony double immunodiffusion test, PCRV was serologically related to a PMV isolate from Oklahoma (PMV-OK.) but not to bean yellow mosaic virus, peanut stripe virus, potato virus Y, watermelon mosaic virus 1, watermelon mosaic virus 2, wheat streak mosaic virus, and zucchini yellow mosaic virus.     

Cowpea Mosaic Virus-Encoded Protease Does Not Recognize Primary Translation Products of M RNAs from Other Comoviruses

The protease encoded by the large (B) RNA segment of cowpea mosaic virus was tested for its ability to recognize the in vitro translation products of the small (M) RNA segment from the comoviruses squash mosaic virus, red clover mottle virus, and cowpea severe mosaic virus (CPsMV, strains Dg and Ark), and from the nepovirus tomato black ring virus. Like M RNA from cowpea mosaic virus, the M RNAs from squash mosaic virus, red clover mottle virus, CPsMV-Dg, and CPsMV-Ark were all translated into two large polypeptides with apparent molecular weights which were different for each virus and even for the two CPsMV strains. Neither the in vitro products from squash mosaic virus, red clover mottle virus, and CPsMV M RNAs nor the in vitro product from tomato black ring virus RNA-2 were processed by the cowpea mosaic virus-encoded protease, indicating that the activity of this enzyme is highly specific.     

Similarity in structure and function of the 3′-terminal region of the four brome mosaic viral RNAs

A 3′-terminal fragment, about 160 nucleotides long, was cleaved by limited nuclease digestion from each of the four RNA components of brome mosaic virus, and purified by two cycles of gel electrophoresis. These fragments accepted tyrosine in reactions catalyzed by wheat germ aminoacyl-tRNA synthetase. Analyses of nuclease digests suggested that the sequences of the fragments from brome mosaic virus RNA 3 and 4 were identical and that the fragments from RNA 1 and 2 differed from that of RNA 4 only in the positions of two and one nucleotides, respectively. A fragment isolated in a similar way from cowpea chlorotic mottle virus was similar in size to the brome mosaic virus RNA fragments, accepted tyrosine in the presence of wheat germ aminoacyl-tRNA synthetase, but had a substantially different nucleotide sequence.     

Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids

Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium-binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca(2+) can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca(2+) ions can induce the capsid contraction and stabilize the native form.     

Genotypic and phenotypic analysis of bromovirus adaptive mutants derived from a single plant

Eight adaptive mutant clones have been made from the total RNA extracted from uninoculated upper leaves of a single cowpea plant exhibiting systemic infection after inoculation with a hybrid cowpea chlorotic mottle bromovirus (CCMV) with the 3a movement protein gene of CCMV replaced by that of cowpea-nonadapted brome mosaic bromovirus (BMV). Sequence and mutational analyses of these clones showed genotypic and phenotypic diversity of the cloned virus population, but all examined clones had the adaptive mutation, A to C at position 776 within the BMV 3a gene, required for the systemic infection of cowpea. The data support the quasispecies model for RNA virus population, and suggest that the maintenance of the adaptive mutation may be due to powerful selection pressure in an infection process.     

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.     

Open reading frame in the minus strand of two plus type RNA viruses

Inspection of the nucleotide sequences of the RNAs complementary to the coat protein mRNAs from two plant viruses with a tripartite genome: alfalfa mosaic virus and brome mosaic virus, showed the presence of open reading frames for 138 and 118 amino acids, respectively. A third virus (cowpea chlorotic mottle virus) from the same family (1) does not show this phenomenon. This suggests that if a protein is coded for by the open reading frames it may be not essential for virus multiplication. Alternatively the open reading frames have no coding function but result from structural requirements of the RNAs.     

Discrimination by gel-diffusion serology of digitaria striate, maize sterile stunt and other rhabdoviruses of Poaceae

Eight rhabdoviruses from grass and cereal hosts and their antisera were used to examine virus relationships by gel-diffusion serology. Nucleocapsid (Nc) preparations from digitaria striate virus (DSV) and maize sterile stunt virus (MSSV) both contained a major protein of c. 52 OOO daltons, and antisera prepared to these readily discriminated related planthopper-transmitted rhabdoviruses. MSSV showed a moderately close relationship to barley yellow striate mosaic virus (BYSMV) when an antiserum prepared to whole virus was used, but the Nc antiserum showed clearer discrimination. Worthern cereal mosaic virus and DSV showed a distant relationship to BYSMV and MSSV. There was no serological relationship between any of these viruses and cereal chlorotic mottle virus, cynodon chlorotic streak virus, festuca leaf streak virus or maize mosaic virus.     

Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.

The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. However, the biological relevance of these two-hybrid interactions to viral positive-strand RNA replication has not been demonstrated. The brome mosaic virus (BMV) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the BMV 1a helicase-like and 2a polymerase-like proteins. We have tested wild-type 1a and 18 insertion mutations of 1a and found a perfect correlation between the in planta phenotypes and their ability to interact with 2a in the two-hybrid system. This finding allowed further characterization of the interaction between and among the BMV viral proteins. Using the two-hybrid assay, we have found that the interaction between the helicase-like region of 1a and the N terminus of 2a is stabilized by the presence of the centrally conserved polymerase-like domain of 2a. We have also identified a novel interaction between the 1a helicase-like protein and itself. Additionally, we have found this interaction in two related tripartite RNA viruses, cowpea chlorotic mottle virus and cucumber mosaic virus. We have demonstrated that this protein-protein interaction is specific to homologous pairings of the protein.     

Nature and specificity of the RNA-protein interaction in the case of the tymoviruses.

Dissociation-reassociation experiments performed with turnip yellow mosaic virus in the presence of various RNAs and polynucleotides were used to investigate the degree of specificity and the contribution of the associated RNA moiety to the stability of TYMV. The results emphasize the importance of strategic cytosine residues spread along the RNA chain. Some insight into the contribution of the protein could be gained from comparison of TYMV and eggplant mosaic virus (EMV), a virus similar to TYMV although its top component contains low molecular mass RNA's able to bind various amino acids. Hydrophobic interactions between protein subunits are less important in EMV than in TYMV, and artificial capsids could be obtained from dissociated EMV coat protein. Whether the capsid is or is not the precursor of the virion in tymovirus morphogenesis is discussed.     

The structure of cucumber mosaic virus and comparison to cowpea chlorotic mottle virus

The structure of cucumber mosaic virus (CMV; strain Fny) has been determined to a 3.2-A resolution using X-ray crystallography. Despite the fact that CMV has only 19% capsid protein sequence identity (34% similarity) to cowpea chlorotic mottle virus (CCMV), the core structures of these two members of the Bromoviridae family are highly homologous. As suggested by a previous low-resolution structural study, the 305-A diameter (maximum) of CMV is approximately 12 A larger than that of CCMV. In CCMV, the structures of the A, B, and C subunits are nearly identical except in their N termini. In contrast, the structures of two loops in subunit A of CMV differ from those in B and C. These loops are 6 and 7 residues longer than the analogous regions in CCMV. Unlike that of CCMV, the capsid of CMV does not undergo swelling at pH 7.0 and is stable at pH 9.0. This may be partly due to the fact that the N termini of the B and C subunits form a unique bundle of six amphipathic helices oriented down into the virion core at the threefold axes. In addition, while CCMV has a cluster of aspartic acid residues at the quasi-threefold axis that are proposed to bind metal in a pH-dependent manner, this cluster is replaced by complementing acids and bases in CMV. Finally, this structure clearly demonstrates that the residues important for aphid transmission lie at the outermost portion of the betaH-betaI loop and yields details of the portions of the virus that are hypothesized to mediate binding to aphid mouthparts.     

Effects of artificial codon changes in the movement protein gene on adaptation of a hybrid bromovirus to cowpea

A hybrid Cowpea chlorotic mottle virus(CCMV) with the movement protein (MP) gene replaced with that of the closely related Brome mosaic virus cannot infect cowpea systemically. Twenty-nine spontaneous mutants from the hybrid CCMV capable of systemic infection in cowpea appeared through biased codon changes that resulted in Lys or Arg at five specific positions in the MP gene. In this study, we report that systemic infection of cowpea with the hybrid CCMV can be achieved by artificial codon changes that do not result in Lys or Arg. We discuss mechanisms that restrict the occurrence of cowpea-adapted mutants in nature.     

Studies on the assembly of a spherical plant virus. I. States of aggregation of the isolated protein

The aggregates formed at equilibrium by purified protein from cowpea chlorotic mottle virus have been characterized on the basis of their sedimentation behaviour and appearance in the electron microscope. Between pH 3.5 and 7.5, at ionic strengths greater than 0.2, most of the protein is found in aggregates sedimenting at either 3 S or 50 S. The 50 S aggregate is identified as the reassembled capsid of cowpea ehlorotic mottle virus. Decreasing the ionic strength favours the formation of multi-shelled particles. Below pH 5.5 single- and multishelled particles predominate, while above this pH most of the protein sediments at 3 S.Varying the temperature from 5 °C to 20 °C has little effect on the equilibrium proportions of aggregates although some real differences can be detected. Ionic strength is not as important a variable as pH in determining which protein forms are present (but increasing ionic strength does result in a steady decrease in the proportion of protein in the multi-layer aggregates). The dependence of the equilibrium upon protein concentration shows that capsid formation is a quasi-crystallization: beyond a certain total protein concentration the concentration of 3 S aggregate remains at this “critical” concentration and all further protein goes into 50 S capsid. In addition to shells and variations upon shells, tubes and hexagonal nets of protein subunits have occasionally been seen with the electron microscope.     

Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana

The complete nucleotide sequence of Chinese rape mosaic virus has been determined. The virus is a member of the tobamovirus genus of plant virus and is able to infect Arabidopsis thaliana (L.) Heynh systemically. The analysis of the sequence shows a gene array that seems to be characteristic of crucifer tobamoviruses and which is slightly different from the one most frequently found in tobamoviruses. Based on gene organization and on comparisons of sequence homologies between members of the tobamoviruses, a clustering of crucifer tobamoviruses is proposed that groups the presently known crucifer tobamovirus into two viruses with two strains each. A name change of Chinese rape mosaic virus to oilseed rape mosaic virus is proposed.Abbreviations 2-ME 2-mercaptoethanol - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - UTR untranslated region - MP movement protein - CP capsid protein - CRMV Chinese rape mosaic virus - TVCV turnip vein clearing virus - PaMMV paprika mild mottle virus - PMMV-I pepper mild mottle virus (Italian isolate) - PMMV-S pepper mild mottle virus (Spanish isolate) - ToMV tomato mosaic virus - TMV tobacco mosaic virus - TMGMV tobacco mild green mosaic virus - ORSV odontoglossum ringspot virus - SHMV sunn hemp mosaic virus - CGMMV cucumber green mottle mosaic virus - ORMV oilseed rape mosaic virus