The synthesis and structure of comovirus capsids.
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Amino Acid Sequence↗
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Similarity in gene organization and homology between proteins of animal picornaviruses and a plant comovirus suggest common ancestry of these virus families.
The amino acid sequences deduced from the nucleic acid sequences of several animal picornaviruses and cowpea mosaic virus (CPMV), a plant virus, were compared. Good homology was found between CPMV and the picornaviruses in the region of the picornavirus 2C (P2-X protein), VPg, 3C pro (proteinase) and 3D pol (RNA polymerase) regions. The CPMV B genome was found to have a similar gene organization to the picornaviruses. A comparison of the 3C pro (proteinase) regions of all of the available picornavirus sequences and CPMV allowed us to identify residues that are completely conserved; of these only two residues, Cys-147 and His-161 (poliovirus proteinase) could be the reactive residues of the active site of a proteinase with analogous mechanism to a known proteinase. We conclude that the proteinases encoded by these viruses are probably cysteine proteinases, mechanistically related, but not homologous to papain.
Amino Acid Sequence↗
Use of enzyme-linked immunosorbent assay for the determination of serological relationship between two comoviruses.
ELISA was used to determine the degree of serological relationship between red clover mottle (RCMV) and broad bean stain (BBSV) viruses and the optimal conditions for the differentiation of the two viruses by ELISA were established. The titres of homologous and heterologous antibodies determined by ELISA were 100- to 200-fold higher as compared with the ring precipitin test. Antigen concentrations of 0.15 and 0.075 ng/ml, respectively, could be detected. The reactions in ELISA depended on the concentration of antigen, of IgG used for coating and of conjugated IgG. In anti-RCMV IgG, the ratio of group-specific to species-specific antibody as determined by ELISA was from 1:1 to 1:2, while in anti-BBSV IgG this ratio was from 1:4--1:8. The differences between homologous and heterologous reactions in ELISA suggested a similar ratio of antigenic determinants in the two viruses.
Antigens, Viral↗
The structure of tobacco ringspot virus: a link in the evolution of icosahedral capsids in the picornavirus superfamily.
BACKGROUND: Tobacco ringspot virus (TRSV) is a member of the nepovirus genus of icosahedral RNA plant viruses that cause disease in fruit crops. Nepoviruses, comoviruses and picornaviruses are classified in the picornavirus superfamily. Crystal structures of comoviruses and picornaviruses and the molecular mass of the TRSV subunit (sufficient to accommodate three beta-barrel domains) suggested that nepoviruses may represent a link in the evolution of the picornavirus capsids from a T = 3 icosahedral virus. This evolutionary process is thought to involve triplication of the capsid protein gene, to encode a three-domain polyprotein, followed by development of cleavage sites in the interdomain linking regions. Structural studies on TRSV were initiated to determine if the TRSV subunit corresponds to the proposed uncleaved three-domain polyprotein. RESULTS: The 3.5 A resolution structure of TRSV shows that the capsid protein consists of three beta-barrel domains covalently linked by extended polypeptides. The order of connectivity of the domains in TRSV confirms the proposed connectivity for the precleaved comovirus and picornavirus capsid polyprotein. Structural differences between equivalent domains in TRSV and comoviruses are confined to the external surface loops, interdomain connecting polypeptides and N termini. The three different domains within TRSV and comoviruses are more closely related at the structural level than the three individual domains within picornaviruses. CONCLUSIONS: The structural results confirm the notion of divergent evolution of the capsid polyproteins of nepoviruses, comoviruses and picornaviruses from a common ancestor. A number of residues were found to be conserved among various nepoviruses, some of which stabilize the quaternary structure of the three domains in the TRSV capsid protein subunit. Two conserved regions were identified on the external surface of TRSV, however, mutational studies will be needed to understand their functional significance. Nepoviruses transmitted by the same nematode species do not share regions with similar amino acid composition on the viral surface.
Amino Acid Sequence↗
Mutational analysis of the genome-linked protein of cowpea mosaic virus.
In this study we have performed a mutational analysis of the cowpea mosaic comovirus (CPMV) genome-linked protein VPg to discern the structural requirements necessary for proper functioning of VPg. Either changing the serine residue linking VPg to RNA at a tyrosine or a threonine or changing the position of the serine from the N-terminal end to position 2 or 3 abolished virus infectivity. Some of the mutations affected the cleavage between the VPg and the 58K ATP-binding protein in vitro, which might have contributed to the lethal phenotype. RNA replication of some of the mutants designed to replace VPg with the related cowpea severe mosaic comovirus was completely abolished, whereas replication of others was not affected or only mildly affected, showing that amino acids that are not conserved between the comoviruses can be critical for the function of VPg. The replicative proteins of one of the mutants failed to accumulate in typical cytopathic structures and this might reflect the involvement of VPg in protein-protein interactions with the other replicative proteins.
Amino Acid Sequence↗
Diverse groups of plant RNA and DNA viruses share related movement proteins that may possess chaperone-like activity.
Amino acid sequences of plant virus proteins mediating cell-to-cell movement were compared to each other and to protein sequences in databases. Two families of movement proteins have been identified, the members of which show statistically significant sequence similarity. The first, larger family (I) encompasses the movement proteins of tobamo-, tobra-, caulimo- and comoviruses, apple chlorotic leaf spot virus (ACLSV) and geminiviruses with bipartite genomes. Thus this family includes viruses which move by two methods, those requiring the coat protein for the cell-to-cell spread (comoviruses) and those not having this requirement (tobamoviruses). The previously unsuspected relationship between the movement proteins of RNA and DNA viruses having no RNA stage in their life cycle (geminiviruses) suggested that their movement mechanisms might be similar. The second, smaller family (II) consists of the movement proteins of tricornaviruses (bromoviruses, cucumoviruses, alfalfa mosaic virus and tobacco streak virus) and dianthoviruses. Alignment of the sequences of family I movement proteins highlighted two motifs, centred at conserved Gly and Asp residues, respectively, which are assumed to be crucial for the movement protein function(s). Screening the amino acid sequence database revealed another conserved motif that is shared by a large subset of family I movement proteins (those of caulimo- and comoviruses, and ACLSV) and the family of cellular 90K heat shock proteins (HSP90). Based on the analogy to HSP90, it is speculated that many plant virus movement proteins may mediate virus transport in a chaperone-like manner.
Amino Acid Sequence↗
Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro.
Tomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domains for the putative NTP-binding protein, VPg, 3C-like protease and a putative RNA-dependent RNA polymerase in its C-terminal region. The N-terminal region of P1, with a coding capacity for a protein (or a precursor) of 67 kDa, has not been characterized. Using partial cDNA clones, it is shown that the 3C-like protease can process the N-terminal region of P1 at a novel cleavage site in vitro, allowing the release of two proteins, X1 (located at the N terminus of P1) and X2 (located immediately upstream of the NTB domain). P1 precursors in which the protease was inactive or absent were not cleaved by exogenously added protease, suggesting that P1 processing was predominantly in cis. Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q(423)/G and Q(620)/G are the X1-X2 and X2-NTB cleavage sites, respectively. The putative X1 protein contains a previously identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature protein released from the N terminus of comovirus P1 polyproteins) and to the corresponding region of other nepovirus P1 polyproteins. These results raise the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruses.
Base Sequence↗
The refined crystal structure of cowpea mosaic virus at 2.8 A resolution.
Comoviruses are a group of plant viruses in the picornavirus superfamily. The type member of comoviruses, cowpea mosaic virus (CPMV), was crystallized in the cubic space group I23, a = 317 A and the hexagonal space group P6(1)22, a = 451 A, c = 1038 A. Structures of three closely similar nucleoprotein particles were determined in the cubic form. The roughly 300-A capsid was similar to the picornavirus capsid displaying a pseudo T = 3 (P = 3) surface lattice. The three beta-sandwich domains adopt two orientations, one with the long axis radial and the other two with the long axes tangential in reference to the capsid sphere. T = 3 viruses display one or the other of these two orientations. The CPMV capsid was permeable to cesium ions, leading to a disturbance of the beta-annulus inside a channel-like structure, suggesting an ion channel. The hexagonal crystal form diffracted X rays to 3 A resolution, despite the large unit cell. The large ( approximately 200 A) solvent channels in the lattice allow exchange of CPMV cognate Fab fragments. As an initial step in the structure determination of the CPMV/Fab complex, the P6(1)22 crystal structure was solved by molecular replacement with the CPMV model determined in the cubic cell.
Amino Acid Sequence↗
Evidence for participation of RNA 1-encoded elicitor in Cowpea mosaic virus-mediated concurrent protection.
The cowpea (Vigna unguiculata) line Arlington, inoculated with Cowpea mosaic virus (CPMV), showed no symptoms, and no infectivity or accumulation of capsid antigen was detected at several days after inoculation. Coinoculation, but not sequential inoculation, of CPMV with similar concentrations of another Comovirus; Cowpea severe mosaic virus (CPSMV), resulted in reduced numbers of CPSMV-induced lesions. This apparent, CPMV-mediated reduction in number of CPSMV-induced infection centers was termed concurrent protection. We report results obtained by inoculating two nearly isogenic cowpea lines derived from a CPMV-susceptible cowpea crossed to Arlington, one line CPMV-susceptible and the other resistant. The CPMV virions B and M, encapsidating genomic RNAs 1 and 2, respectively, were extensively purified by gradient centrifugation. In the CPMV-resistant cowpea, either CPMV or CPMV B affected concurrent protection against CPSMV and against two distinct non-Comoviruses: Cherry leafroll virus and Southern bean mosaic virus. Adding CPMV M to the inoculum did not enhance CPMV-B-mediated protection. CPMV B was ineffective in protecting CPMV-susceptible cowpea. We postulate that CPMV-mediated concurrent protection is elicited in CPMV-resistant cowpea by a CPMV RNA-1-encoded factor and acts to reduce accumulation or spread of CPMV and certain coinoculated challenging viruses in or from the inoculated cell. Coinoculated CPMV did not protect CPMV-resistant cowpea against Tomato bushy stunt virus or Cucumber mosaic virus.
Comovirus↗
Diversity among isolates of squash mosaic virus.
cDNA clones of RNA-2 of two isolates of squash mosaic virus (SqMV) were constructed and sequenced, revealing 87% sequence similarity. In Northern blot hybridization analyses, DNA probes made from these clones defined two SqMV hybridization subgroups. This grouping was verified by reciprocal hybridizations of purified RNA from five SqMV isolates, as probed with cDNA made from a member of each subgroup. Comparison of the RNA-2 sequence among the two SqMV isolates, and the reported sequence of other comoviruses, showed that SqMV constitutes one of four major branches in a phylogenetic tree of the genus. Analysis of the terminal noncoding sequences showed that although potentially similar folding patterns may form, neither nucleotide sequence nor secondary structural elements are highly conserved among comoviruses. In vitro translation products from purified RNA-1 of each subgroup (encoding the viral proteases) were found to process the polyprotein generated by in vitro translation of purified RNA-2 from either subgroup.
Amino Acid Sequence↗
Preparation and specificity of antibodies against coat proteins of broad bean stain virus.
Mouse polyclonal antibodies were prepared against broad bean stain virus (BBSV, Comovirus group) and its coat protein subunits, large (L) and small (S) protein. These subunits were less immunogenic than native virus. Antibodies against L protein (Anti-L) and native virus (Anti-BBSV) did not react in immunoblots with S protein, but Anti-BBSV antibody reacted with S protein in plate-trapped antigen ELISA. Anti-S antibodies did not react with the related red clover mottle comovirus (RCMV), but Anti-L and Anti-BBSV antibodies reacted with RCMV similarly to BBSV. We assume that all the linear epitopes of the BBSV S protein are hidden in the native virions and the antigenic similarity between BBSV and RCMV is based mainly on their common linear L-specific epitopes.
Animals↗
Characterization of monoclonal antibodies against broad bean stain and red clover mottle viruses.
Seven monoclonal antibodies (MoAbs) against red clover mottle comovirus (RCMV) and/or broad bean stain comovirus (BBSV) were characterized and used for epitope comparison of both viruses. All tested MoAbs, exclusively of IgG class, were directed to detergent-stable epitopes (metatopes and cryptotopes). Two of them were species-specific, while five others cross-reacted with both viruses to different extent. On the basis of the results of competitive ELISA, we assume different mutual position of common linear epitopes in RCMV and BBSV. They are more closely "packed up" on BBSV, while the BBSV-specific metatope is markedly dominant. The investigation of other RCMV and BBSV isolates by use of our MoAbs confirmed close relationship between both viruses and even showed that it is difficult to determine unambiguously the species of these isolates using immunochemical methods only.
Antibodies, Monoclonal↗
Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: infectious transcripts and complete nucleotide sequence of RNA 1.
Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5'-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5'-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.
Amino Acid Sequence↗
Molecular cloning and sequence analysis of a segment from Andean potato mottle virus B RNA encoding the putative RNA polymerase.
Andean potato mottle virus (APMV), an endemic South American comovirus, has a bipartite genome consisting of two plus-strand RNA molecules (M and B RNA). We have cloned the 3' half of the B RNA and identified the complete sequence of the putative APMV RNA polymerase. The RNA polymerase gene is part of a large polyprotein-encoding open reading frame. The putative, mature RNA polymerase, as deduced by comparison with the related cowpea mosaic virus (CPMV), type member of the comovirus group, is 703 amino acids long and shows a large degree of similarity with CPMV and other RNA polymerases.
Amino Acid Sequence↗
Adaptation of positive-strand RNA viruses to plants.
The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection of plant hosts. Genome sequence comparisons of these plant RNA viruses have revealed that most of them are genetically related to animal cell-infecting counterparts; this led to the concept of "superfamilies". Comparison of genetic maps of representative plant and animal viruses belonging to the same superfamily (e.g. cowpea mosaic virus [CPMV] versus picornaviruses and tobacco mosaic virus versus alphaviruses) have revealed genes in the plant viral genomes that appear to be essential adaptations needed for successful invasion and spread through their plant hosts. The best studied example represents the "movement protein" gene that is actively involved in cell-to-cell spread of plant viruses, thereby playing a key role in virulence and pathogenesis. In this paper the host adaptations of a number of plant viruses will be discussed, with special emphasis on the cell-to-cell movement mechanism of comovirus CPMV.
Adaptation, Physiological↗
The nucleotide sequence of the coat protein genes of satsuma dwarf virus and naval orange infectious mottling virus.
The sequence of the 3'-terminal 4320 and 2409 nucleotides were determined for RNA2 of satsuma dwarf virus (SDV) and navel infectious mottling virus (NIMV). Both sequences contained a part of a long open reading frame which encodes larger and smaller coat proteins (CPs) at the 3'-terminus followed by a 3'non-coding region upstream of a poly (A) tail. Amino acid sequence identity for larger and smaller CPs ranged 81-84% and 68-78%, respectively, among SDV, NIMV and the previously sequenced citrus mosaic virus (CiMV). No significant sequence similarity was found between the CPs of SDV or NIMV and those of the como-, nepo- or other viruses. The nucleotide sequence identity of the 3' non-coding region of RNA2 were 68%-78% among SDV, CiMV and NIMV. These results suggest that SDV, CiMV and NIMV are distinct, though related, viruses. They may be assigned as members of the new genus, which is close to the genera of Comovirus and Nepovirus.
Amino Acid Sequence↗
Nucleotide sequence of a Japanese isolate of Squash mosaic virus. Brief report.
The complete nucleotide sequence of the RNA-1 of Squash mosaic virus (SqMV) was determined using a Japanese isolate (Y-SqMV). The sequence consisted of 5865 nucleotides excluding the poly (A) at the 3' terminus and contained a single long open reading frame with a coding capacity for a protein of Mr209971. Analysis of the deduced amino acid sequence suggested a genomic organization typical of comoviruses. The nucleotide sequence of the RNA-2 of Y-SqMV was also determined and compared with the SqMV isolates from the United States. The larger and smaller capsid protein (CP) coding region was compared to those of K-SqMV and Z-SqMV, which represent two subgroups of SqMV. The larger CP gene of Y-SqMV showed 93.0% and 88.0% identities with those of K-SqMV and Z-SqMV, respectively at the nucleotide level. The smaller CP gene of Y-SqMV was 94.1% and 88.4% identical with those of K-SqMV and Z-SqMV. The results suggested that the Japanese SqMV isolate (Y-SqMV) is distinct from those in the United States, and might represent a third subgroup.
Comovirus↗
The Bean pod mottle virus proteinase cofactor and putative helicase are symptom severity determinants.
Full-length infectious cDNA clones were constructed from the genomic RNAs of three distinct strains (K-G7, K-Ha1 and K-Ho1) of the comovirus Bean pod mottle virus (BPMV). Whereas K-G7, a subgroup I strain, and K-Ha1, a subgroup II strain produce mild mottling, the reassortant strain K-Ho1 (RNA1(I) + RNA2(II)) induces necrotic primary lesions on inoculated leaves of soybean and severe systemic leaf mottling and blistering. Pseudorecombinants of all possible combinations of transcripts were generated and tested for symptom production. Only soybean plants inoculated with combinations having RNA1 derived from the severe strain K-Ho1, regardless of the origin of RNA2, induced severe symptoms, indicating that symptom severity maps to RNA1. Experiments with chimeric RNA1 constructs indicated that the coding regions of the protease co-factor (Co-pro) and the C-terminal half of the putative helicase (Hel) are determinants of symptom severity. Symptom severity correlated well with higher accumulation of viral RNA, but neither the Co-pro nor Hel protein could be demonstrated as a suppressor of RNA silencing.
Base Sequence↗